Plasmids and phages for homologous recombination and methods of use

ABSTRACT

Lambda phages that can be used to introduce recombineering functions into host cells are disclosed. Also disclosed are plasmids that can be used to confer recombineering functions to a variety of strains of  E. coli  and to other bacteria, including  Salmonella, Pseudomonas, Cyanobacteria, Spirochaetes . These plasmids and phages can be isolated in vitro and can be used to transform bacterial cells, such as gram negative bacteria.

PRIORITY CLAIM

This is a continuation of U.S. patent application Ser. No. 12/688,764,filed on Jan. 15, 2010, which is a divisional of U.S. patent applicationSer. No. 11/134,795, filed on May 20, 2005, which issued as U.S. Pat.No. 7,674,621, and which claims the benefit of U.S. ProvisionalApplication No. 60/655,729, filed Feb. 22, 2005, U.S. ProvisionalApplication No. 60/653,259, filed Feb. 14, 2005, and U.S. ProvisionalApplication No. 60/573,504, filed May 21, 2004. All of the priorapplications are incorporated by reference herein in their entirety.

FIELD

This application relates to recombinant DNA technology, specifically toplasmids and phages of use for introducing homologous recombinationfunctions into host cells.

BACKGROUND

Concerted use of restriction endonucleases and DNA ligases allows invitro recombination of DNA sequences. The recombinant DNA generated byrestriction and ligation may be amplified in an appropriatemicroorganism such as E. coli, and used for diverse purposes includinggene therapy. However, the restriction-ligation approach has twopractical limitations: first, DNA molecules can be precisely combinedonly if convenient restriction sites are available; second, becauseuseful restriction sites often repeat in a long stretch of DNA, the sizeof DNA fragments that can be manipulated are limited, usually to lessthan about 20 kilobases.

Homologous recombination, generally defined as an exchange of homologoussegments anywhere along a length of two DNA molecules, provides analternative method for engineering DNA. In generating recombinant DNAwith homologous recombination, a microorganism such as E. coli, or aeukaryotic cell such as a yeast or vertebrate cell, is transformed withan exogenous strand of DNA. The center of the exogenous DNA contains thedesired transgene, whereas each flank contains a segment of homologywith the cell's DNA. The exogenous DNA is introduced into the cell withstandard techniques such as electroporation or calciumphosphate-mediated transfection, and recombines into the cell's DNA, forexample with the assistance of recombination-promoting proteins in thecell.

In generating recombinant DNA by homologous recombination, it is oftenadvantageous to work with short linear segments of DNA. For example, amutation may be introduced into a linear segment of DNA using polymerasechain reaction (PCR) techniques. Under proper circumstances, themutation may then be introduced into cellular DNA by homologousrecombination. Such short linear DNA segments can transform yeast, butsubsequent manipulation of recombinant DNA in yeast is laborious. It isgenerally easier to work in bacteria, but linear DNA fragments do notreadily transform bacteria (due in part to degradation by bacterialexonucleases). Accordingly, recombinants are rare, require specialpoorly-growing strains (such as RecBCD− strains) and generally requirethousands of base pairs of homology.

Recently, a method for homologous recombination, termed “recombineering”has made it possible to clone nucleic acids in specific strains of E.coli using homologous recombination. However, the number of strains ofE. coli that can be used in this method are limited. Thus, methods ofintroducing recombineering functions into other strains of E. coli areneeded. In addition, methods of introducing these functions into otherbacteria, including other gram negative bacteria, are also needed.

SUMMARY

Recombineering utilizes the recombination functions encoded by lambdoidbacteriophages to efficiently catalyze homologous recombination in vivobetween DNA sequences with homologies as short as 35 bases.Recombineering provides methods to clone and modify genes on plasmids,on BACs, on the chromosome of enteric bacteria, and on bacteriophage λwithout the necessity of restriction enzymes or DNA ligase.Recombineering also allows rapid and precise in vivo manipulation ofDNA.

Disclosed herein are plasmids that can be used to confer recombineeringfunctions to a variety of cells, including strains of E. coli,Salmonella, Pseudomonas, Cyanobacteria, and Spirochaetes, amongstothers. These mobilizable plasmids can be manipulated in vitro and canbe used to transform bacterial cells, such as gram negative bacteria.These plasmids include an origin of replication specific for thebacterial cell(s) of interest, a de-repressible promoter, and a nucleicacid encoding a single-stranded binding protein such as Beta. Inadditional embodiments, the plasmids include a nucleic acid encoding Exoand/or Gam. In one example, the plasmid includes an origin ofreplication and a lambda genome having DNA encoding functional Beta andoptionally Exo, and Gam, or functional fragments or variants thereof,operably linked to a de-repressible promoter (such as, but not limitedto, the λ P_(L) promoter). In one example, the plasmids include anorigin of replication, a nucleic acid encoding a selectable marker, anucleic acid encoding a promoter operably linked to nucleic acidsequence encoding a repressor that specifically binds a de-repressiblepromoter, and the de-repressible promoter operably linked to a nucleicacid encoding Beta and a terminator 3′ of the nucleic acid encoding asingle-stranded binding protein. Bacterial host cells transformed withthe vector are capable of performing homologous recombination.

Lambda phages that can be used to introduce recombineering functionsinto host cells are also disclosed herein. These phages include ambermutations in an essential gene(s) and include a selectable marker. Inthis manner, the phage will enter the lytic cycle in a host cell thatincludes a suppressor of the amber mutation and cause host cell death.However, the phage will be able to lysogenize in cells that do notinclude the suppressor mutation(s). In one example, the phage includes arepressor that binds a P_(L) promoter, a promoter operably linked to anucleic acid encoding a heterologous nucleic acid sequence, P_(L), and anucleic acid encoding Beta operably linked to P_(L), a nucleic acidencoding P, a nucleic acid encoding O, and a nucleic acid encoding Cro.At least two of the nucleic acids (genes) encoding P, the nucleic acidencoding O, and the nucleic acid encoding Cro include an amber codon.Thus, at least two of P, O, and Cro proteins are not produced when thelambda phage is introduced into a suppressor minus host cell, but areproduced in host cells that include appropriate tRNA suppressors. Inthis manner lytic phage in high yields can be produced in host cellsthat include the appropriate tRNA suppressors.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a schematic diagram showing classical recombinant technology,and FIG. 1B is a schematic diagram showing “recombineering” usinghomologous recombination, as disclosed herein.

FIG. 2 is a schematic diagram of the genetic map of lambda phage,showing the position of the major genes in a wild-type lambda nucleicacid.

FIG. 3 is a schematic diagram of the plasmid pSIM4 pUC rex<>amp.

FIGS. 4A-F are the nucleic acid sequence (SEQ ID NO: 2) of pSIM4 (seeFIG. 3), which encodes cI (SEQ ID NO: 10), Gam (SEQ ID NO: 11), Bet (SEQID NO: 12) and Exo (SEQ ID NO: 13) and Bla (Amp^(R)) (SEQ ID NO: SEQ IDNO: 15). The amino acid sequence for each of the proteins is shown belowthe DNA sequence of pSIM4 (SEQ ID NO: 2) identified with its letters.Since the genes are transcribed from the sequence shown in the figurefrom right to left the proteins are shown in this orientation, and thenumber of the amino acid is shown at the left edge. Each protein startswith M (for methionine) and above that M in the DNA sequence is the nameof the gene (protein) Gam, Beta or Exo (SEQ ID NOs: 11-13). In thesequence shown in the panels of this figure, the coding sequence of DNAis the bottom strand. Thus, the start codon is “GTA” on the bottom butis read right to left as “ATG.” Exo starts M T P D I I (the first aminoacids of SEQ ID NO: 13), Beta starts M S T A L (the first amino acids ofSEQ ID NO: 12), Gam starts M N A Y Y (the first amino acids of SEQ IDNO: 11). The nucleic acid encoding the phage genes from pSIM4, pSIM6 andpSIM8 is set forth as SEQ ID NO: 9.

FIG. 5 is a schematic diagram of the plasmid pSIM 5-pSC101 rex<>cat.

FIGS. 6A-G is the nucleotide sequence of pSIM5 (SEQ ID NO: 3). The phagegenes from pSIM5 are set forth in the nucleotide sequence of SEQ ID NO:8 (the amino acid sequences of Beta, Gam and Exo are set forth as SEQ IDNOs: 11-13). The sequence of the CAT drug cassette is shown in SEQ IDNO: 14, and the amino acid sequence of Repts is shown in SEQ ID NO: 16.The amino acid sequence of c1857 is shown in SEQ ID NO: 10, and theamino acid sequence of the Orf of pSIM5 is set forth as SEQ ID NO: 17.

FIG. 7 is a schematic diagram of plasmid pSIIVI6-pSC101 rex<>amp (SEQ IDNO: 4).

FIGS. 8A-F are the nucleic acid sequence (SEQ ID NO: 4) ofpSIIVI6-pSC101 rex<>amp (see FIG. 6) and the polypeptides encoded bythis plasmid (SEQ ID NOs: 10-13, 15, 16 and 17). The phage genes ofpSIM6 are shown in the nucleotide sequence set forth as SEQ ID NO: 9.

FIG. 9 is a schematic diagram of plasmid pSIM8-pBBR1 rex<>amp (SEQ IDNO: 6).

FIGS. 10A-G are the nucleic acid sequence (SEQ ID NO: 6) of plasmidpSIM8-pBBR1 rex<>amp and the polypeptides encoded by this plasmid (SEQID NOs: 10-13, 15, 18). The phage genes of pSIM8 are shown in thenucleotide sequence set forth as SEQ ID NO: 9.

FIG. 11 is a schematic diagram of plasmid pSIM2 with rex<>cat.

FIGS. 12A-E are the nucleotide sequence of pSIM2 (SEQ ID NO: 1) and theencoded polypeptides (SEQ ID NOs: 10-14). The phage genes of pSIM2 areset forth as SEQ ID NO: 8.

FIG. 13 is a schematic diagram of plasmid pSIM7 with rex<>cat (SEQ IDNO: 5).

FIG. 14A-G are the nucleotide sequence of pSIM7 (SEQ ID NO: 5); thenucleotide sequence of the phage genes are set forth as SEQ ID NO: 8.Polypeptides encoded by the plasmid are set forth as SEQ ID NOs: 10-13,14, and 18.

FIGS. 15A-G are the nucleotide sequence of pSIM9 (SEQ ID NO:7). Theamino acid sequences of the encoded polypeptides (SEQ ID NOs: 10-14, 14,18 and 19) are also shown.

FIG. 16A is a schematic diagram of a lambda prophage without the N-kilregion and wherein a selectable marker is inserted into the prophage rexgenes. FIG. 16B is a schematic diagram of the generation of a plasmidusing the prophage shown in FIG. 16A by the method of recombineering(retrieval by gap repair of a linear vector containing terminalhomologies to the target). FIGS. 16C-16D are schematic diagrams of thecreation of a PCR product including a pBR322-type origin of replication(FIG. 16C) and the replacement of an origin on a plasmid with anotherorigin of interest (FIG. 16D).

FIGS. 17A-B are the lambda nucleic acid sequence (SEQ ID NO: 24) showingthe rex genes. The sequence numbers are the same as in the lambdagenomic sequence. The protein sequence of RexA (SEQ ID NO: 25 and RexB(SEQ ID NO: 26) is shown. Note that the amino acid sequence of RexA andRexB each begin with a methioine (M). In the figure the sequences areread from the initiating methionine (at the N-terminus) from right toleft.

FIGS. 18A-B are the nucleic acid sequence of the rexAB<>tetRA whereinthe tet genes (SEQ ID NO: 29) replace the rexAB genes (SEQ ID NO: 25,SEQ ID NO: 26). The flanking sequence of lambda is used to illustratethe exact sequence replacement. The sequence numbers at the top left arethe same as in the lambda genomic sequence. The amino acid sequences oftetA (SEQ ID NO: 28) and tetR (SEQ ID NO: 27) are shown.

FIG. 19 is the lambda nucleic acid sequence of the S gene (SEQ ID NO:30) encoding the S protein (SEQ ID NO: 31). The sequence numbers are thesame as in the lambda genomic sequence.

FIGS. 20A-B are the sequence of S<>tetRA wherein the S gene is replacedby the tetRA genes. The sequence numbers at the top left are the same asin the lambda genomic sequence. The amino acid sequences of tetA (SEQ IDNO: 13) and tetR (SEQ ID NO: 14) are shown.

FIG. 21 is the nucleic acid sequence (SEQ ID NO: 21) encoding the S.typhimurium galK protein (galK−, SEQ ID NO: 20).

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. If only one strand of each nucleic acid sequence is shown, thecomplementary strand is understood as included by any reference to thedisplayed strand, if appropriate in context.

The Sequence Listing is submitted as an ASCII text file[4239-68523-06_Sequence_Listing.txt, Jun. 26, 2013, 105 KB], which isincorporated by reference herein.

In the accompanying sequence listing:

SEQ ID NO: 1 is the nucleic acid sequence of pSIM2.

SEQ ID NO: 2 is the nucleic acid sequence of pSIM4.

SEQ ID NO: 3 is the nucleic acid sequence of pSIM5.

SEQ ID NO: 4 is the nucleic acid sequence of pSIM6.

SEQ ID NO: 5 is the nucleic acid sequence of pSIM7.

SEQ ID NO: 6 is the nucleic acid sequence of pSIM8.

SEQ ID NO: 7 is the nucleic acid sequence of pSIM9.

SEQ ID NO: 8 is the nucleic acid sequence of the phage genes of pSIM2,pSIM5, pSIM7 and pSIM9 plasmids.

SEQ ID NO: 9 is the nucleic acid sequence of the phage genes of pSIM4,pSIM6 and pSIM8 plasmids.

SEQ ID NO: 10 is the amino acid sequence of cI857.

SEQ ID NO: 11 is the amino acid sequence of Gam.

SEQ ID NO: 12 is the amino acid sequence of Beta.

SEQ ID NO: 13 is the amino acid sequence of Exo.

SEQ ID NO: 14 is the amino acid sequence of the CAT drug cassette, usedin pSIM2, pSIM5, pSIM7 and pSIM9.

SEQ ID NO: 15 is the amino acid sequence of the Amp drug cassette, usedin pSIM4, pSIM6 and pSIM8.

SEQ ID NO: 16 is the amino acid sequence of Repts of pSIM5 and pSIM6.

SEQ ID NO: 17 is the amino acid sequence of the Orf of pSIM5 and pSIM6.

SEQ ID NO: 18 is the amino acid sequence of Rep of pSIM7 and pSIM8.

SEQ ID NO: 19 is the amino acid sequence of the replication gene TrfAtsfrom pSIM9.

SEQ ID NO: 20 is the amino acid sequence of galK.

SEQ ID NO: 21 is the nucleic acid sequence encoding galK.

SEQ ID NO: 22 is the nucleic acid sequence of an oligonucleotide.

SEQ ID NO: 23 is the nucleic acid sequence of an oligonucleotide.

SEQ ID NO: 24 is the nucleic acid sequence encoding RexAB.

SEQ ID NO: 25 is the amino acid sequence of RexA.

SEQ ID NO: 26 is the amino acid sequence of RexB.

SEQ ID NO: 27 is the amino acid sequence of tetR.

SEQ ID NO: 28 is the amino acid sequence of tetA.

SEQ ID NO: 29 is the nucleic acid sequence encoding tetRA.

SEQ ID NO: 30 is the nucleic acid sequence encoding S.

SEQ ID NO: 31 is the amino acid sequence of S.

SEQ ID NO: 32 is the nucleic acid sequence of an oligonucleotide.

SEQ ID NO: 33 is the nucleic acid sequence of an oligonucleotide.

SEQ ID NO: 34 is the nucleic acid sequence of an oligonucleotide.

SEQ ID NO: 35 is the nucleic acid sequence of an oligonucleotide.

DETAILED DESCRIPTION I. Abbreviations

Amp: ampicillin

BAC: bacterial artificial chromosome

Bp: base pairs

Cat: chloramphenicol acetyl-transferase

Ini: initiation

λ: lambda

Ori: origin of replication

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

Antibiotic Resistance Cassette:

A nucleic acid sequence encoding a selectable marker which confersresistance to that antibiotic in a host cell in which the nucleic acidis translated. Examples of antibiotic resistance cassettes include, butare not limited to: kanamycin, ampicillin, tetracycline,chloramphenicol, neomycin, hygromycin and zeocin.

Attachment Site (att):

A specific site for recombination that occurs on either a phage or achromosome. An attachment site on lambda is termed “attP,” while anattachment site of a bacterial chromosome is “attB.” Integrase mediatedrecombination of an attP site with an attB site leads to integration ofthe λ prophage in the bacterial chromosome.

Bacterial Artificial Chromosome (BAC):

Bacterial artificial chromosomes (BACs) have been constructed to allowthe cloning of large DNA fragments in E. coli, as described in O'Conneret al., Science 244:1307-12, 1989; Shizuya et al., Proc. Natl. Acad.Sci. U.S.A. 89:8794-7, 1992; Hosoda et al., Nucleic Acids Res.18:3863-9, 1990; and Ausubel et al., eds., Current Protocols InMolecular Biology, John Wiley & Sons (c) 1998 (hereinafter Ausubel etal., herein incorporated in its entirety). This system is capable ofstably propagating mammalian DNA over 300 kb. In one embodiment, a BACcarries the F replication and partitioning systems that ensure low copynumber and faithful segregation of plasmid DNA to daughter cells. Largegenomic fragments can be cloned into F-type plasmids, making them of usein constructing genomic libraries.

Beta:

The 28 kDa lambda Beta ssDNA binding polypeptide (and nucleic acidencoding lambda beta) involved in double-strand break repair homologousrecombination. DNA encoding Beta (bet) and polypeptide chains havinglambda Beta activity are also referred to herein as bet. See Examples 1and 14 and references therein for further information. The lambda Betaprotein binds to single-stranded DNA and promotes renaturation ofcomplementary single-strand regions of DNA (see also Karakousis et al.,J. Mol. Biol. 276:721-733, 1998; Li et al., J. Mol. Biol. 276:721-733,1998; Passy et al., PNAS 96:4279-4284, 1999).

Functional fragments and variants of Beta include those variants thatmaintain their ability to bind to ssDNA and mediate the recombinationfunction of lambda Beta as described herein, and in the publicationsreferenced herein. It is recognized that the gene encoding Beta may beconsiderably mutated without materially altering the ssDNA bindingfunction or homologous recombination function of lambda Beta. First, thegenetic code is well-known to be degenerate, and thus different codonsencode the same amino acids. Second, even where an amino acid mutationis introduced, the mutation may be conservative and have no materialimpact on the essential functions of lambda Beta. See Stryer,Biochemistry 3rd Ed., (c) 1988. Third, part of the lambda Betapolypeptide chain may be deleted without impairing or eliminating itsssDNA binding protein function, or its recombination function. Fourth,insertions or additions may be made in the lambda Beta polypeptidechain—for example, adding epitope tags—without impairing or eliminatingits essential functions (see Ausubel et al., 1997, supra).

Biolistics:

Insertion of DNA into cells using DNA-coated micro-projectiles. Alsoknown as particle bombardment or microparticle bombardment. The approachis further described and defined in U.S. Pat. No. 4,945,050, which isherein incorporated by reference.

cDNA (Complementary DNA):

A piece of DNA lacking internal, non-coding segments (introns) andregulatory sequences that determine transcription. cDNA may besynthesized in the laboratory by reverse transcription from messengerRNA extracted from cells.

Cro:

A very small protein, the wild-type form of which includes 66 aminoacids. The protein includes a single domain which contains a DNA-bindinghelix-turn-helix. The Cro protein binds the operator sites (O_(L) andO_(R)) of lambda. It interferes with the binding of cI, which is arepressor that also binds to the operator sites of lambda. Transcriptionof the Cro and cI is regulated by the cI protein. Specifically, in theabsence of cI proteins, the Cro gene can be transcribed, while in thepresence of cI proteins, only the cI gene is transcribed. At highconcentrations of cI, transcriptions of both genes are repressed.Temperature sensitive mutations of cI have been described, such ascI857. In these temperature sensitive forms, the function of cI isinhibited at high temperatures (such as when the temperature isincreased from 37° C. to 42° C.). The sequence and functions of Cro andcI are well known, and are described, for example, in Ptashne et al., AGenetic Switch, Third Edition, Phage Lambda Revisited, Cold SpringHarbor Press, Cold Spring Harbor, New York, 2004, which is incorporatedherein by reference. The structure and sequence of lambda, including Croand cI can also be found on the internet.

De-Repressible Promoter:

When a repressor is bound to a de-repressible promoter, transcription issubstantially decreased as compared to transcription from thede-repressible promoter in the absence of the repressor. By regulatingthe binding of the repressor, such as by changing the environment, therepressor is released from the de-repressible promoter and transcriptionincreases.

One specific, non-limiting example of a de-repressible promoter is theP_(L) promoter, which is regulated by the repressor cI. P_(L) is notactivated by an activator, and thus is not an inducible promoter. An“activatable promoter” is a promoter wherein binding of an activator tothe promoter increases transcription from the promoter. The arabinosepromoter, pBAD is not a simple de-repressible promoter; arabinoseinactivates the repressor AraC and converts it to an activator. Thus,pBAD is an activatable promoter.

In one embodiment, the de-repressible promoter is a temperaturesensitive de-repressible promoter. A temperature sensitivede-repressible promoter is a promoter that is de-repressed only at aspecified temperature, or range of temperatures. In one embodiment, byincreasing the temperature, the repressor is released from the promoter,or can no longer bind to the promoter with a high affinity, andtranscription is increased from the promoter. One specific, non-limitingexample is the induction of P_(L) promoter activity by increasing thetemperature of the cell using cI87. Increased temperature inactivatesthe temperature-sensitive repressor cI, allowing genes that are operablylinked to the P_(L) promoter to be expressed at increased levels. One ofskill in the art can readily identify a de-repressible promoter.

In one embodiment, a de-repressible promoter is auto-regulated. Onespecific, non-limiting example of an auto-regulated de-repressiblepromoter is P_(L). If only one copy of a gene encoding cI is present,yet many copies of the P_(L) promoter are present, expression of cI isupregulated such that transcription is blocked from any of the P_(L)promoters.

Double-Strand Break Repair Recombination:

A type of homologous recombination exemplified by the lambdarecombination proteins Exo, Beta and Gam, and shared by numerous otherrecombinase systems. A double-strand break is the initiation point forconcerted action of recombination proteins. Typically, an exonucleasedegrades processively from the 5′ ends of these break sites, and ssDNAbinding polypeptide binds to the remaining 3′ single-strand tail,protecting and preparing the recessed DNA for homologous strand invasion(Szostak et al., Cell 33:25-35, 1983; Little, J. Biol. Chem.242:679-686, 1967; Carter et al., J. Biol. Chem. 246:2502-2512, 1971;Lindahl et al., Science 286:1897-1905, 1999). Examples of ssDNA bindingpolypeptides which bind to either ssDNA and/or dsDNA with 3′ overhangsand promote double-strand break repair recombination include lambdaBeta, RecT of E. coli, Erf of phage p22, and Rad52 in various eukaryoticcells including yeast and mammalian cells.

Electrocompetent:

Cells capable of macromolecular uptake upon treatment withelectroporation.

Electroporation:

A method of inducing or allowing a cell to take up macromolecules byapplying electric fields to reversibly permeabilize the cell walls.Various methods and apparatuses used are further defined and describedin: U.S. Pat. No. 4,695,547; U.S. Pat. No. 4,764,473; U.S. Pat. No.4,946,793; U.S. Pat. No. 4,906,576; U.S. Pat. No. 4,923,814; and U.S.Pat. No. 4,849,089, all of which are herein incorporated by reference.

Eukaryotic Cell:

A cell having an organized nucleus bounded by a nuclear membrane. Theseinclude lower organisms such as yeasts, slime molds, and the like, aswell as cells from multicellular organisms such as invertebrates,vertebrates and mammals. They include a variety of tissue types, suchas, but not limited to, endothelial cell, smooth muscle cell, epithelialcell, hepatocyte, cells of neural crest origin, tumor cell,hematopoietic cell, immunologic cell, T cell, B cell, monocyte,macrophage, dendritic cell, fibroblast, keratinocyte, neuronal cell,glial cell, adipocyte, myoblast, myocyte, chondroblast, chondrocyte,osteoblast, osteocyte, osteoclast, secretory cell, endocrine cell,oocyte, and spermatocyte. These cell types are described in standardhistology texts, such as McCormack, Introduction to Histology, (c) 1984by J. P. Lippincott Co.; Wheater et al., eds., Functional Histology, 2ndEd., (c) 1987 by Churchill Livingstone; Fawcett et al., eds., Bloom andFawcett: A Textbook of Histology, (c) 1984 by William and Wilkins, allof which are incorporated by reference in their entirety. In onespecific, non-limiting example, a eukaryotic cell is a stem cell, suchas an embryonic stem cell.

Exo:

The exonuclease of lambda (and the nucleic acid encoding the exonucleaseprotein) involved in double-strand break repair homologousrecombination. See Example 1 and references therein for furtherdescription.

Exogenous:

The term “exogenous” as used herein with reference to nucleic acid and aparticular cell refers to any nucleic acid that does not originate fromthat particular cell as found in nature. Thus, a non-naturally-occurringnucleic acid is considered to be exogenous to a cell once introducedinto the cell. Nucleic acid that is naturally occurring also can beexogenous to a particular cell. For example, an entire chromosomeisolated from a cell of subject X is an exogenous nucleic acid withrespect to a cell of subject Y once that chromosome is introduced intoY's cell.

Extrachromosomal:

Not incorporated into the chromosome or chromosomes of a cell. In thecontext of nucleic acids, extrachromosomal indicates a DNAoligonucleotide that is not covalently incorporated into the chromosomeor chromosomes of a cell. Intrachromosomal refers to material such as anoligonucleotide that is incorporated into the chromosome or chromosomesof a cell, such as a DNA oligonucleotide covalently incorporated intothe chromosomal DNA of a cell.

Flanking:

In the sequence “A-B-A”, nucleic acid sequence “A” flanks nucleic acidsequence “B.” In one specific, non-limiting example, nucleic acidsequence “A” is located immediately adjacent to nucleic acid “B.” Inanother specific, non-limiting example, an linker sequence of not morethan 500 nucleotides is between each copy of “A” and “B,” such as alinker sequences of about 200, about 100, about 50, or about 10nucleotides in length. Nucleotide sequences “A” and “B” can be of anylength. “Adjacent” refers to a first nucleic acid sequence next to asecond amino acid sequence. Thus, in the sequence A-B-C, A is 5′ to Band adjacent to B. However, A is 5′ to C but is not adjacent to C. B is3′ of A and 5′ of C; B is adjacent to both A and C and is flanked by Aand C.

Gam:

A lambda protein (and nucleic acid encoding Gam) involved indouble-strand break repair homologous recombination. It is believed toinhibit cellular nuclease activity such as that encoded by the recBCDand sbcC system of E. coli. See Examples 1, 7 and 14 and referencestherein for further description. Gam function, when expressed in thecell, is extremely toxic to the cell, and prevents growth. For thisreason tight controls over its expression are always required. Asdescribed herein, P_(L) and cI 857 are able to regulate Gam expression.

Functional fragments and variants of Exo and Gam: As discussed for Beta(see “Functional Fragments And Variants Of Beta”), it is recognized thatgenes encoding Exo or Gam may be considerably mutated without materiallyaltering their function, because of genetic code degeneracy,conservative amino acid substitutions, non-critical deletions orinsertions, etc. Unless the context makes otherwise clear, the termlambda Exo, Exo or lambda exonuclease are all intended to include thenative lambda exonuclease, and all fragments and variants of lambdaexonuclease.

Gene:

A nucleic acid encoding a protein product. In a specific non-limitingexample, a gene includes at least one expression control sequence, suchas a promoter, enhancer or a repressor. In another specific,non-limiting example, a gene includes at least one intron and at leastone exon.

Homology Arm:

Nucleotides at or near 5′ or 3′ end of a polynucleotide which areidentical or similar in sequence to the target nucleic acid in a cell,and capable of mediating homologous recombination with the targetnucleic acid. Homology arms are also referred to as homologous arms. Inone embodiment, a homology arm includes at least 20 bases of a sequencehomologous to a nucleic acid of interest. In another embodiment, thehomology arm includes at least 30 base pairs of a sequence homologous toa nucleic acid of interest. In yet another embodiment, a homology armincludes at least 40 base pairs of a sequence homologous to a nucleicacid of interest. In a further embodiment, a homology arm includes fromabout 50 to about 100 base pairs of a sequence homologous to a nucleicacid of interest.

Homologous Recombination:

An exchange of homologous polynucleotide segments anywhere along alength of two nucleic acid molecules. In one embodiment, two homologoussequences are 100% identical. In another embodiment, two homologoussequences are sufficiently identical such that they can undergohomologous recombination. Specific, non-limiting examples of homologoussequences are nucleic acid sequences that are at least 95% identical,such as about 99% identical, about 98% identical, about 97% identical,or about 96% identical.

Host Cell:

A cell that is used in lab techniques such as DNA cloning to receiveexogenous nucleic acid molecules. In one embodiment a host cell is usedto maintain or allow the reproduction of a vector, or to facilitate themanipulation of nucleic acid molecules in vitro. A host cell can be aprokaryotic or a eukaryotic cell. In one embodiment, a host cell is agram negative bacterial cell. In another embodiment, a host cell is agram positive host cell.

HVJ-Mediated Gene Transfer:

A method of macromolecular transfer into cells using inactivatedhemagglutinating virus of Japan and liposomes, as described in Morishitaet al., J. Clin. Invest. 91:2580-2585, 1993; Morishita et al., J. Clin.Invest. 94:978-984, 1994; which are herein incorporated by reference.

Inducible Promoter:

A promoter whose activity can be increased by the binding of an inducerto the promoter. Examples of inducible promoters abound in nature, and abroad range of environmental or hormonal changes may activate or repressthem. One specific example of an inducible promoter is pBAD.

Initiation Site or Replication:

The site on a nucleic acid sequence wherein of DNA replication occurs.For example, a bacterial origin of replication is the site on thebacterial DNA wherein DNA replication starts. For example, an initiationsite can be a ColE1 initiation site (Tomizawa et al., PNAS 74:1865-69,1077) or an E. coli or B. subtilis oriC (see Seitz et al., EMBO Reports2:1003-1006, 2001).

Intron:

An intragenic nucleic acid sequence in eukaryotes that is not expressedin a mature RNA molecule. Introns of the present disclosure includefull-length intron sequences, or a portion thereof, such as a part of afull-length intron sequence.

Isolated:

An “isolated” biological component (such as a nucleic acid or protein)has been substantially separated or purified away from other biologicalcomponents in the cell of the organism in which the component naturallyoccurs, i.e., other chromosomal and extra-chromosomal DNA and RNA, andproteins. Thus, nucleic acids and proteins that have been “isolated”include nucleic acids and proteins purified by standard purificationmethods. The term also embraces nucleic acids and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids.

Knockout:

Inactivation of a gene such that a functional protein product cannot beproduced. A conditional knockout is a gene that is inactivated underspecific conditions, such as a gene that is inactivated in atissue-specific or a temporal-specific pattern. A conditional knockoutvector is a vector including a gene that can be inactivated underspecific conditions. A conditional knockout transgenic animal is atransgenic animal including a gene that can be inactivated in atissue-specific or a temporal-specific manner.

Linear Plasmid Vector:

A DNA sequence (1) containing a bacterial plasmid origin of replication,(2) having a free 5′ and 3′ end, and (3) capable of circularizing andreplicating as a bacterial plasmid by joining its free 5′ and 3′ ends.Examples of linear plasmid vectors include the linearized pBluescriptvector and linearized pBR322 vectors described herein.

Linker Region:

DNA which connects flanking regions of a plasmid. The linker region caninclude multi-cloning sites which contain recognition sites for specificrestriction endonucleases and transcriptional terminator-sequence.Linker regions can be ligated to the ends of DNA fragments prepared bycleavage with some other enzyme. A linker region can also have uniquerestriction endonuclease sites at the location of the start and stopcodon to ligate the 5′ flanking region, as well as the 3′ flankingregion to the nucleic acid of the linker. In particular, the linkerregion provides recognition sites, i.e., the “multicloning sites,” forinserting the nucleic acid cassette which contains a specific nucleicsequence to be expressed. These recognition sites may be a restrictionendonuclease site in the linker, such as BamHI, EcoRI, HindIII, ClaI,NotI, XmaI, BglII, PacI, XhoI, NheI, SfiI, which are only examples andnot meant to be limiting. The multicloning site permits easy insertionof expression nucleic acid elements such as promoters, nucleic acidsencoding selectable markers or therapeutic genes, etc. For example, themulticloning site in pBluescript KS+ provides 17-23 unique restrictionsites useful in inserting expression elements or previously constructednucleic acid cassettes.

Lipofection:

The process of macromolecular transfer into cells using liposomes. SeeU.S. Pat. No. 5,651,981, which is herein incorporated by reference.

Mini-Lambda:

A derivative of lambda (λ) wherein most of the viral lytic genes,including those required for replication and lysis, are deleted. Amini-lambda maintains the Red functions (Beta, Exo, and Gam) forhomologous recombination and maintains the integration/excisionfunctions (for example, att, integrase (int) and excisionase (xis)) toinsert and excise its DNA from the chromosome.

Nucleic Acid:

A deoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, including known analogs of natural nucleotidesunless otherwise indicated.

Oligonucleotide (Oligo):

A single-stranded nucleic acid ranging in length from 2 to about 500bases, for example, polynucleotides that contain at least 20 or 40nucleotides (nt). Oligonucleotides are often synthetic but can also beproduced from naturally occurring polynucleotides.

Operably Linked:

A first nucleic acid sequence is operably linked with a second nucleicacid sequence when the first nucleic acid sequence is placed in afunctional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if thepromoter affects the transcription or expression of the coding sequence.Generally, operably linked DNA sequences are contiguous and, wherenecessary to join two protein-coding regions, in the same reading frame.

Operator Site (O_(R) and O_(L)):

A nucleic acid sequence found in lambda, repressor binding at thesesites reduces transcription of N and Cro from P_(L) and P_(R),respectively. The right complete operator region (O_(R)) can besubdivided into three operators, O_(R)1, O_(R)2, and O_(R)3. Similarly,the left complete operator can also be subdivided into three operators.The repressor, cI, binds to both O_(R)1 and O_(R)2, and binding toO_(R)2 stimulates the transcription of the cI mRNA. The repressor, cI,binds to the three operator regions in the order of: cI binds mosttightly to O_(R)1, next to O_(R)2, and lastly to O_(R)3. The binding ofCro and cI to this region differs in terms of the order of interaction;Cro binds most tightly to O_(R)3, next to O_(R)2, and lastly to O_(R)1.Similarly, Cro and cI bind to O_(L)1, O_(L)2, and O_(L)3 with differentaffinities.

Origin of Replication (ori):

A nucleotide sequence at which DNA synthesis for the purpose ofreplicating the nucleic acid sequence begins. This is generally termedan “ori” site. Circular bacterial chromosomes generally have a singleori site, whereas there are many ori sites on each eukaryoticchromosome. This term includes replicons, which as used herein refers toa genetic element that behaves as an autonomous unit during DNAreplication. In bacteria, the chromosome functions as a single replicon,whereas eukaryotic chromosomes contain hundreds of replicons in series.

The ori site of plasmids can allow replication in one or more bacterialspecies, such as a gram negative or a gram positive species. Forexample, an on can allow replication in one or more of E. Coli,Yersinia, or Salmonella. Specific, non-limiting examples of an ori are aColE1 origin and its derivatives, a pSC101 origin and its derivatives, apBBR1 origin and its derivatives, and a RK2 origin and its derivatives.In one specific example, a ColE1 origin of replication is described inTomizawa et al., Proc. Natl. Acad. Sci. (PNAS) 74:1865-69, 1977 (such as-420 to −613 base pairs (upstream) of the initiation site of ColE1replication).

A “conditional origin of replication” refers to an origin of replicationthat requires the presence of a functional transacting factor (e.g., areplication factor) in a prokaryotic host cell. Conditional origins ofreplication encompass temperature-sensitive replicons such as reppSC101.

Operator Sequence:

A specific nucleic acid sequence capable of interacting with a specificrepressor, thereby controlling the function of genes in adjacentcistrons and regulator genes. In general, a regulator gene is a genewhose primary function is to control the rate of synthesis of theproducts of other distant genes. The regulator gene controls thesynthesis of a protein repressor, which inhibits the action of anoperator gene and thus turns off the operon it controls. The repressorusually is present in small amounts. It may possess two sites, one ofwhich can attach to the operator and one of which can bind an effectormolecule. In one embodiment, once a repressor is bound to an effectormolecule, the repressor changes shape and cannot attach to the operator.In another embodiment (such as for λ cI857) heat itself can inactivateor denature the intact repressor so that it does not attach to theoperator. An operon is a unit of nucleic acid sequence consisting of oneor more cistrons that function coordinately under the control of anoperator sequence.

Thus, the repressor is a protein, synthesized by a regulator gene, thatbinds to an operator locus and blocks transcription of that operon. Therepressor causes repression of transcription. When de-repressed,transcription and/or translation are increased.

Phage-Based Recombination Systems:

Bacteria such as E. coli encode their own homologous recombinationsystems, which are used in repair of DNA damage and to maintain afunctional chromosome. The viruses or phages that inhabit bacteria oftencarry their own recombination functions. Phage λ carries the Redrecombination system. These phage systems can work with the bacterialrecombination functions or independently of them. It should be notedthat a prophage is the latent state of a phage in a lysogenic bacterium.“Induction” is the process that converts a prophage into a phage.

P_(L) promoter: The major leftward promoter of bacteriophage lambda.Once the lambda DNA is incorporated into the bacterial chromosome,transcription from this promoter is substantially repressed by the cIrepressor. Upon inactivation of the cI repressor, for example by heatshock of a temperature sensitive mutant, transcription from the P_(L)promoter is de-repressed, leading to expression of lambda genes. SeeSambrook et al., “Bacteriophage Lambda Vectors,” Chapter 2 in MolecularCloning: a Laboratory Manual, 2nd Ed., (c) 1989 (hereinafter Sambrook etal.); Stryer, “Control of Gene Expression in Procaryotes,” Chapter 32 inBiochemistry 3rd Ed., pp. 799-823, (c) 1988 (hereinafter Stryer); andCourt and Oppenheim, pp. 251-277 in Hendrix et al. eds., Lambda II, ColdSpring Harbor Lab Press, (c) 1983 (hereinafter Court and Oppenheim).

P_(R) Promoter:

The major rightward promoter of bacteriophage lambda. Once the lambdaDNA is incorporated into the bacterial chromosome, transcription fromthis promoter is substantially repressed by the cI repressor. Uponinactivation of the cI repressor, for example by heat shock of atemperature sensitive mutant, transcription from the P_(R) promoter isde-repressed, leading to expression of lambda genes. See Sambrook etal., “Bacteriophage Lambda Vectors,” Chapter 2 in Molecular Cloning: aLaboratory Manual, 2nd Ed., (c) 1989 (hereinafter Sambrook et al.);Stryer, “Control of Gene Expression in Procaryotes,” Chapter 32 inBiochemistry 3rd Ed., pp. 799-823, (c) 1988.

Plasmid:

A plasmid is a construction of genetic material designed to directtransformation of a targeted cell. Plasmids include a construction ofextrachromosomal genetic material, usually of a circular duplex of DNAwhich can replicate independently of chromosomal DNA. A plasmidgenerally contains multiple genetic elements positional and sequentiallyoriented with other necessary genetic elements such that the nucleicacid in a nucleic acid cassette can be transcribed and when necessarytranslated in the transfected cells. Plasmids include nucleic acids thatare DNA derived from a plasmid vector, cosmids, or phagemids wherein oneor more fragments of nucleic acid may be inserted or cloned which encodefor particular genes of interest. The plasmid can have a linear orcircular configuration.

Plasmids generally contain one or more unique restriction sites. Inaddition, a plasmid can confer some well-defined phenotype on the hostorganism which is either selectable or readily detected. Thus, theplasmid can include an expression cassette, wherein a polypeptide isencoded. Expression includes the efficient transcription of an insertedgene, nucleic acid sequence, or nucleic acid cassette with the plasmid.Expression products can be proteins, polypeptides or RNA.

In one embodiment, when a circular plasmid is transferred into abacterial cell, it can be an autonomously replicating, extra-chromosomalDNA molecule, distinct from the normal bacterial genome and nonessentialfor bacterial cell survival under nonselective conditions. The term“persistent expression” as used herein refers to introduction of genesinto the cell together with genetic elements which enable episomal(extra-chromosomal) replication and/or maintenance of the geneticmaterial in the cell. This can lead to apparently stable transformationof the cell without the integration of the novel genetic material intothe chromosome of the host cell.

A plasmid can also introduce genetic material into chromosomes of thetargeted cell where it integrates and becomes a permanent component ofthe genetic material in that cell. Gene expression after stableintroduction can permanently alter the characteristics of the cell andits progeny arising by replication leading to stable transformation.

Polynucleotide:

A double-stranded or single-stranded nucleic acid sequence of anylength. Therefore, a polynucleotide includes molecules which are 15, 50,100, 200 nucleotides long (oligonucleotides) and also nucleotides aslong as a full length cDNA.

Probes and Primers:

A nucleic acid probe comprises an isolated nucleic acid attached to adetectable label or reporter molecule. Typical labels includeradioactive isotopes, ligands, chemiluminescent agents and enzymes.Methods for labeling and guidance in the choice of labels appropriatefor various purposes are discussed, e.g., in Sambrook et al. (1989) andAusubel et al. (1997).

Primers are short nucleic acids, preferably DNA oligonucleotides aboutfifteen nucleotides or more in length. Primers may be annealed to acomplementary target DNA strand by nucleic acid hybridization to form ahybrid between the primer and the target DNA strand. The 3′ hydroxyl endof the primer may be then extended along the target DNA strand throughthe use of a DNA polymerase enzyme. Primer pairs (one on either side ofthe target nucleic acid sequence) can be used for amplification of anucleic acid sequence, e.g., by the polymerase chain reaction (PCR) orother nucleic-acid amplification methods known in the art.

Methods for preparing and using probes and primers are described, forexample, in Sambrook et al. (1989), Ausubel et al. (1987). PCR primerpairs can be derived from a known sequence, for example, by usingcomputer programs intended for that purpose such as Primer (Version 0.5,© 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).Under appropriate conditions, the specificity of a particular probe orprimer increases with its length. Thus, in order to obtain greaterspecificity, probes and primers may be selected that comprise 20, 25,30, 35, 40, 50 or more consecutive nucleotides of related cDNA or genesequence.

Prokaryote:

Cell or organism lacking a membrane-bound, structurally discrete nucleusand other subcellular compartments.

Prokaryotic Transcription Termination Sequence:

A nucleic acid sequence which is recognized by the RNA polymerase of aprokaryotic host cell and results in the termination of transcription.There are two types of terminators, one requires the Rho protein incombination with RNA polymerase at certain Rho-dependent sequences whilethe other is intrinsic and depends on sequence alone to stop polymerase.Prokaryotic intrinsic termination sequences commonly include a GC-richregion that has a twofold symmetry followed by an AT-rich sequence(Stryer, supra). A commonly used prokaryotic termination sequence is therRNA operon termination sequence. A variety of termination sequences areknown to the art and may be employed in nucleic acid constructsincluding the T_(INT), T_(L1), T_(L2), T_(L3), T_(R1), T_(R2), T_(6S)termination signals derived from the bacteriophage lambda, andtermination signals derived from bacterial genes such as the trp gene ofE. coli (see Stryer, supra).

Promoter:

An array of nucleic acid control sequences which direct transcription ofa nucleic acid. A promoter includes necessary nucleic acid sequencesnear the start site of transcription, such as in the case of apolymerase II type promoter, a TATA element. Enhancer and repressorelements can be located adjacent or distal to the promoter, and can belocated as much as several thousand base pairs from the start site oftranscription. Examples of promoters include, but are not limited to,the λ P_(L) and P_(R) promoters, the SV40 promoter, the CMV promoter,the β-actin promoter, and tissue-specific promoters. A hybrid promoteris a promoter that directs transcription of a nucleic acid in botheukaryotic and prokaryotic cells. One specific, non-limiting example ofa hybrid promoter is a PGK-EM7 promoter. Another specific, non-limitingexample of a hybrid promoter is PGK-Tnf.

Recombinant Nucleic Acid Molecule:

A nucleic acid molecule which is comprised of segments of DNA joinedtogether by means of molecular biological techniques, or that isproduced from such a molecule, such as following replication of aplasmid. Strands of a DNA molecule are said to have 5′ ends and 3′ endsbecause mononucleotides are reacted to make oligonucleotides in a mannersuch that the 5′ phosphate of one mononucleotide pentose ring isattached to the 3′ oxygen of its neighbor in one direction via aphosphodiester linkage. In a duplex DNA molecule, each strand has a 5′and a 3′ end. An end of an oligonucleotide referred to as the “5′ end”if its 5′ phosphate is not linked to the 3′ oxygen of a mononucleotidepentose ring and as the “3′ end” if its 3′ oxygen is not linked to a 5′phosphate of a subsequent mononucleotide pentose ring. As used herein, anucleic acid sequence, even if internal to a larger oligonucleotide,also may be said to have 5′ and 3′ ends. In either a linear or circularDNA molecule, discrete elements are referred to as being “upstream” or5′ of the “downstream” or 3′ elements relative to a reference nucleicacid sequence of a fixed element in DNA that has a polarized directionin a single strand. This terminology reflects the fact thattranscription proceeds by making RNA in a 5′ to 3′ fashion along one ofthe DNA strands (such as an unmethylated strand of DNA). The promoterand enhancer elements which direct transcription of a linked gene aregenerally located 5′ relative to a strand transcribed into RNA, orupstream of the coding region. However, enhancer elements can exerttheir effect even when located 3′ of the promoter element and the codingregion. Transcription termination and polyadenylation signals arelocated 3′ or downstream of the coding region.

Regulatory Element:

A genetic element which controls some aspect of the expression ofnucleic acid sequences. For example, a promoter is a regulatory elementwhich facilitates the initiation of transcription of an operably linkedcoding region. Other regulatory elements are splicing signals,polyadenylation signals, termination signals, etc. Regulatory elementsinclude “promoter” and “enhancer” elements. Promoters and enhancersconsist of short arrays of DNA sequences that interact specifically withcellular proteins involved in transcription (Maniatis et al., Science236:1237, 1987). Promoter and enhancer elements have been isolated froma variety of eukaryotic sources including genes in yeast, insect andmammalian cells and viruses (analogous control elements, i.e.,promoters, are also found in prokaryotes). The selection of a particularpromoter and enhancer depends on what cell type is to be used to expressthe protein of interest. Some eukaryotic promoters and enhancers have abroad host range while others are functional in a limited subset of celltypes. A regulatory element can be “endogenous” or “heterologous.” An“endogenous” regulatory element is one which is naturally linked with agiven gene in the genome. A “heterologous” regulatory element is onewhich is placed in juxtaposition to a gene by means of geneticmanipulation; the regulatory element is not naturally found adjacent toa reference nucleic acid sequence, such as in a wild-type organism.

Restriction Endonucleases and Restriction Enzymes:

Bacterial enzymes, each of which cut double-stranded DNA at or near aspecific nucleotide sequence. A “restriction site” is a deoxyribonucleicacid sequence at which one or more specific restriction endonucleasescleave the molecule.

Selectable Marker:

A nucleic acid (or a protein encoded by the nucleic acid) which can beused to identify a cell, such as a host cell, of interest. Selectablemarkers include but are not limited to: (1) nucleic acid segments thatencode products which provide resistance against otherwise toxiccompounds (e.g., antibiotics); (2) nucleic acid segments that encodeproducts which are otherwise lacking in the recipient cell (e.g., tRNAgenes, auxotrophic markers); (3) nucleic acids that encode productswhich suppress the activity of a gene product; (4) nucleic acids thatencode products which can be readily identified (such as phenotypicmarkers such as B-galactosidase, green fluorescent protein (GFP), andcell surface proteins); (5) nucleic acids that bind products which areotherwise detrimental to cell survival and/or function; (6) nucleicacids that otherwise inhibit the activity of any of the nucleic acidsdescribed in Nos. 1-5 above (e.g., antisense oligonucleotides); (7)nucleic acids that bind products that modify a substrate (e.g.restriction endonucleases); (8) nucleic acids that can be used toisolate a desired molecule (e.g. specific protein binding sites); (9) aspecific nucleotide sequence which can be otherwise non-functional(e.g., for PCR amplification of subpopulations of molecules); and/or(10) DNA segments, which when absent, directly or indirectly confersensitivity to particular compounds.

In one example, the nucleic acid encodes an enzymatic activity thatconfers the ability to grow in medium lacking what would otherwise be anessential nutrient (e.g., the TRP1 gene in yeast cells). In anotherexample, a selectable marker can confer resistance (or sensitivity) toan antibiotic or drug upon the cell in which the selectable marker isexpressed. In a further example, the selectable marker can also be usedto confer a particular phenotype upon a host cell.

When a host cell must express a selectable marker to grow in selectivemedium, the marker is said to be a positive selectable marker (e.g.,antibiotic resistance genes which confer the ability to grow in thepresence of the appropriate antibiotic). Selectable markers can also beused to select against host cells containing a particular gene (e.g.,the sacB gene which, if expressed, kills the bacterial host cells grownin medium containing 5% sucrose); selectable markers used in this mannerare referred to as negative selectable markers or counter-selectablemarkers.

Sequence Identity:

The relatedness between two nucleic acid sequences, or two amino acidsequences is expressed in terms of the similarity between the sequences,otherwise referred to as sequence identity or homology. Sequenceidentity is frequently measured in terms of percentage identity (orsimilarity or homology); the higher the percentage, the moresimilar/homologous are the two sequences.

Methods of alignment of sequences for comparison are well-known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Bio. 48:443, 1970; Pearson and Lipman, Methods in Molec. Biology24:307-331, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins andSharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research16:10881-90, 1988; Huang et al., Computer Applications in BioSciences8:155-65, 1992; and Pearson et al., Methods in Molecular Biology24:307-31, 1994. Altschul et al. (Nature Genet., 6:119-29, 1994)presents a detailed consideration of sequence alignment methods andhomology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-410, 1990) is available from several sources,including the National Center for Biological Information (NBCI,Bethesda, Md.) and on the internet, for use in connection with thesequence analysis programs blastp, blastn, blastx, tblastn and tblastx.It can be accessed at the NCBI website, together with a description ofhow to determine sequence relatedness using this program.

Homologs of lambda Beta, Exo and Gam, and ssDNA binding proteins (suchas the Herpes simplex virus single-stranded binding protein) typicallypossess at least some (for example, at least 60%) sequence identitycounted over limited or full-length alignment with the amino acidsequence of the protein being evaluated (that is, lambda Beta, Exo orGam, or other ssDNA binding protein). Homologs of other proteins, suchas P22 Erf, RecT, and Rad52, or the Herpes virus single ICP8 strandedbinding protein and UL12 exonuclease can also be identified. Homologs ofa protein can be identified, for example, using the NCBI Blast 2.0,gapped blastp set to default parameters. For comparisons of amino acidsequences of greater than about 30 amino acids, the Blast 2 sequencesfunction is employed using the default BLOSUM62 matrix set to defaultparameters, (gap existence cost of 11, and a per residue gap cost of 1).When aligning short peptides (fewer than around 30 amino acids), thealignment should be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). Proteins with even greater similarity to thereference sequence will show increasing percentage identities whenassessed by this method, such as at least 70%, at least 75%, at least80%, at least 90%, at least 95%, at least 98%, or at least 99% sequenceidentity. When less than the entire sequence is being compared forsequence relatedness, homologs will typically possess at least 75%sequence identity over short windows of 10-20 amino acids, and maypossess sequence identities of at least 85% or at least 90% or 95%depending on their similarity to the reference sequence. Methods fordetermining sequence identity over such short windows are described atthe NCBI web site

One of skill in the art will appreciate that these sequence identityranges are provided for guidance only; it is entirely possible thatstrongly significant homologs or other variants could be obtained thatfall outside of the ranges provided.

Single-Stranded DNA (ssDNA) and Double-Stranded DNA (dsDNA):

ssDNA is DNA in a single polynucleotide chain; the DNA bases are notinvolved in Watson-Crick base pairing with another polynucleotide chain.dsDNA involves two or more complementary polynucleotide chains, in whichthe two polynucleotide chains are at least partially Watson-Crickbase-paired to each other. Note that a segment of DNA may be partiallyssDNA and partially dsDNA, for example if there are gaps in onepolynucleotide chain of a segment of dsDNA, or there are 5′ or 3′overhangs. ssDNA and dsDNA may contain nucleotide analogs, non-naturallyoccurring or synthetic nucleotides, biotin, or epitope or fluorescenttags. ssDNA or dsDNA may be labeled; typical labels include radioactiveisotopes, ligands, chemiluminescent agents and enzymes.

Suppressor of Nonsense Mutations:

Nonsense mutations are examples of conditional mutations—in a strainlacking a nonsense suppressor (suppressor minus or sup°), the mutationcauses premature termination of protein synthesis, but in a strain withan appropriate nonsense suppressor (sup), functional protein can bemade. If a phage vector contains nonsense mutations in a gene essentialfor lysis, it will only be able to reproduce in a bacterial host with anappropriate nonsense suppressor.

One type of a nonsense mutation is an amber mutation. An ambersuppressor inserts an amino acid at only an amber (UAG) codon. A supB orsupC suppressor inserts an amino acid at UAA and UAG codons. Anothertype of nonsense mutation is an ochre mutation. In one example, if aphage mutant with an amber mutation at position #50 of an essential geneinfects a sup° host, no functional protein will be made and the phagewill not reproduce. If the phage infects a supD host (which includes atRNA that recognizes the amber codon and inserts a serine), a serinewill be inserted at position #50 of the protein; if the phage infects asupE host, a glutamine will be inserted at position #50 of the protein;if the phage infects a supF host, a tyrosine will be inserted atposition #50 of the protein; and so on. If the amino acid inserted atthis position results in a misfolded, truncated, or otherwisenonfunctional protein, the phage will not reproduce on the suppressorcontaining host. However, if the amino acid inserted at this site yieldsa functional protein, the phage will reproduce. A number of suppressorsare known:

original suppressor codon amino-acid name anticodon anticodon suppressedinserted supB UUG UUA UAA, UAG Gln supC GUA UUA UAA, UAG Tyr supD CGACUA UAG Ser supE CUG CUA UAG Gln supF GUA CUA UAG Tyr supG UUU UUAUAA, UAG Lys supM UAA, UAG Tyr supN UAA, UAG Lys supO UAA, UAG Tyr supPUAG Leu supU CCA UCA UGA Trp

Transcriptional Terminator Element:

A nucleotide sequence that functions to stop transcription of an RNApolymerase without additional factors is an intrinsic terminator. Thisintrinsic sequence can be located within the linker region, or after anucleic acid encoding Bet, Gam or Exo, but can be located at other sitesin the plasmid. These sequences ensure transcription of the nucleic acidsequence does not read through into other functional regions of theplasmid. The term “transcription” or “transcribe” refers to the processby which RNA molecules are formed upon DNA templates by complementarybase pairing. This process is mediated by RNA polymerase. In oneembodiment, the terminators are derived from E. coli rrnB operon.Additional terminators include the λ terminators in the P_(L) and P_(R)operon, such as T_(L3), T_(L4) and T_(L2).

Transformation, Transduction and Transfection:

The introduction of foreign DNA into prokaryotic or eukaryotic cells.Transformation of prokaryotic cells may be accomplished by a variety ofmeans known to the art including the treatment of host cells with CaCl₂to make competent cells, electroporation, etc. Transfection ofeukaryotic cells can be accomplished by a variety of means known to theart including calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, liposome fusion, lipofection,protoplast fusion, retroviral infection and biolistics.

Upstream:

Refers to nucleic acid sequences that precede the codons that aretranscribed into an RNA of interest, or to a nucleic acid sequences 5′of a nucleic acid of interest. Similarly, “downstream” refers to nucleicacid sequences that follow codons that are transcribed into a RNA ofinterest, or to nucleic acid sequences 3′ of a nucleic acid of interest.

Vector:

Nucleic acid molecules that transfer DNA segment(s) from one cell toanother. A “vector” is a type of “nucleic acid construct.” The term“nucleic acid construct” includes circular nucleic acid constructs suchas plasmid constructs, cosmid vectors, etc. as well as linear nucleicacid constructs (e.g., λ phage constructs, PCR products). The nucleicacid construct may include expression signals such as a promoter and/oran enhancer (in such a case, it is referred to as an expression vector).An “expression vector” is a recombinant DNA molecule containing adesired coding sequence and appropriate nucleic acid sequences necessaryfor the expression of the operably linked coding sequence in aparticular host organism. Nucleic acid sequences necessary forexpression in prokaryotes usually include a promoter and can alsoinclude an operator. Eukaryotic cells are known to utilize promoters,enhancers, and termination and polyadenylation signals.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods and examplesare illustrative only and not intended to be limiting.

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

There exists a need in the art for methods of precisely and efficientlyaltering predetermined endogenous genetic sequences by homologousrecombination in vivo, in a variety of gram negative bacterial cells.Plasmids, phages and methods are disclosed herein for cloning DNAmolecules in gram negative bacterial cells using homologousrecombination mediated by lambda recombinases.

Recombineering

FIG. 1A depicts a classical genetic engineering protocol to modify atarget on a BAC clone with a cassette and compares the classicaltechnology with the recombineering technology disclosed herein that usesspecial phage recombination functions. In general, there are many stepsrequired for classical engineering, and the final product cannot beengineered as precisely as by the new recombineering technology. Anadvance in the recombineering methodology is the use of phagerecombination functions that generate recombination products usinghomologies of 50 bases (or less). Note that the target homologies inFIG. 1A and FIG. 1B are represented by the striped boxes. In the methodoutlined in FIG. 1A, those boxes must be at least 500 base pairs long,whereas in the method outlined in FIG. 1B, they can be about 40 to about50 base pairs long. In several examples, the homologies are about 30 to100 bases long, or from about 40 to about 100 bases in length.

One method of recombineering involves transforming a gram negativebacterial cell of interest (such as, but not limited to, an E. colicell) with a plasmid including an origin of replication, and a lambdagenome having DNA encoding functional Beta and optionally Exo, and Gam,or functional fragments or variants thereof, operably linked to the ade-repressible promoter (such as, but not limited to, the P_(L)promoter). De-repression of the de-repressible promoter (for example,the induction of transcription from the P_(L) promoter by inactivationof cI) induces expression of Exo, Bet and Gam. In some embodimentsde-repression may be selectively activated for this purpose. Anothermethod of recombineering involves the introduction of a phage or a phageincluding the P_(L) promoter operably linked to a nucleic acid encodingBeta, and optionally Exo and Gam, or functional fragments or variantsthereof.

In recombineering, a polynucleotide which is homologous to a target DNAsequence (capable of undergoing homologous recombination with the targetDNA sequence) is introduced into the cell. Cells in which homologousrecombination has occurred are either selected or found by directscreening of cells. In particular embodiments, the nucleic acidintroduced into the cell may be single-stranded DNA, double-strandedDNA, or DNA with 5′ overhangs. These methods are disclosed, for example,in PCT Publication No. WO 02/14495 A2 and in U.S. Patent Publication No.US-2003-0224521-A1 (both of which incorporated by reference herein intheir entirety).

Briefly, the recombineering methodology utilizes recombination functions(such as phage recombination functions) under control of ade-repressible promoter to generate recombination products usinghomologies of at least 20 base pairs. Thus, in one embodiment,recombineering uses a cell including Beta under the control of ade-repressible promoter. In a specific, non-limiting example, expressionof Beta alone (without Exo and Gam) is under the control of thede-repressible promoter (e.g. the nucleic acid encoding Beta is operablylinked to the de-repressible promoter). In another embodiment,expression of Beta, in addition to Gam and/or Exo, is under the controlof the de-repressible promoter. In further embodiments, the geneencoding ICP8, RecT, P22 Erf, or Rad52 is operably linked to ade-repressible promoter. In yet another embodiment, DNA bound to a Betaprotein is introduced into a host cell.

In recombineering, phage recombination functions can be used tointroduce recombination into a target nucleic acid sequence in a hostcell. The host cell can be prokaryotic. In specific non-limitingexamples, the host cell is any gram negative bacterial cell, including,but not limited to, E. coli or S. typhimurium. The target can be on thechromosome, or can be on an extra-chromosomal element. In severalspecific, non-limiting examples, the target nucleic acid can be includedin a plasmid, a bacterial artificial chromosome (BAC), a yeastartificial chromosome, a cosmid or a vector, including but not limitedto a viral vector. In one specific non-limiting example, recombinationis induced in a BAC strain or a BAC DNA is introduced into straincarrying recombination functions.

The length of the homologous sequence can be varied. In severalembodiments, the homology is at least 20, at least 25, at least 30, atleast 40, at least 50, at least 75 or at least 100 nucleotides inlength. However, larger regions of homology can also be utilized. Thus,in one embodiment, between about 20 and about 1,000 nucleotides ofhomologous sequence is utilized, between about 30 and about 1,000nucleotides of homologous sequence is utilized. In one specific,non-limiting example, the ssDNA is about 20, about 25, about 30, about40, about 50, about 75 or about 100 nucleotides in length. In oneembodiment, the homologous nucleic acid is a single-stranded nucleicacid. In another embodiment, the homologous nucleic acid is adouble-stranded nucleic acid. Double-stranded nucleic acids includemolecules that are completely double-stranded, as well as nucleic acidmolecules that have a 5′ or a 3′ overhang.

A single-stranded nucleic acid or double-stranded nucleic acid includingsufficient homology to the target sequence is introduced into the hostcell. “Sufficient homology” is any region of sufficient identity to thetarget sequence such that recombination can occur. In severalembodiments, sufficient homology includes a sequence of at least 20nucleotides in length, wherein at most five, at most three, at most two,at most one nucleotide, or no nucleotides differ from the target nucleicacid sequence. In additional embodiments, sufficient homology includes asequence of at least 25 nucleotides in length, wherein at most five, atmost three, at most two, at most one nucleotide, or no nucleotidesdiffer from the target nucleic acid sequence. Similarly, sufficienthomology can readily be determined for a nucleic acid of at least 30, atleast 40, at least 50, or at least 100 nucleotides in length.

If the single-stranded nucleic acid or double-stranded nucleic aciddiffers from the target nucleic acid, these differences can be clustered(i.e. at one area in the target nucleic acid) or can be scattered in thesequences (for example two nucleotide differences from the targetsequence, wherein each difference is located at different areas in thesequence). In another embodiment, sufficient homology includes about a100%, 99%, 98%, or 97% sequence identity between the homolgous nucleicacid (e.g., the single-stranded or the double-stranded nucleic acid) andthe target nucleic acid sequence. In another specific, non-limitingexample, sufficient homology includes at least 90% sequence identitybetween the single-stranded or double-stranded nucleic acid and thetarget nucleic acid, such as nucleic acid sequences that are at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% identical. It should benoted that a homologous nucleic acid sequence can differ from a targetnucleic acid by substitutions, deletions and/or additions ofnucleotides. In another embodiment, the single-stranded nucleic acid (ordouble-stranded nucleic acid) is labeled, such as with a biotinylatednucleotide, a methylated nucleotide, or a DNA adduct.

The homologous nucleic acid (e.g., the single-stranded nucleic acid ordouble-stranded nucleic acid) can be introduced into the host cell byany means known to one of skill in the art. In one embodiment, the hostcell is deficient in mismatch repair, such as a cell that can repairmismatched nucleotides at a reduced frequency as compared to a wild-typecell (such as at least 50%, 60%, 70%, 80%, 90%, 95%, 98%, or 99%reduction in mismatch repair). In one specific, non-limiting example,mismatch repair is reduced at least 90% as compared to a wild-type cell.A host cell deficient for mismatch repair can include a mutation in anucleic acid sequence encoding a protein involved in mismatch repair,such that the protein has reduced function (or its function iseliminated). In several embodiments, the function of one or moremismatch repair proteins is decreased at least 80%, such as at least90%, 95%, 96%, 97%, 98%, 99%, or is completely absent in the host celldeficient for mismatch repair as compared to a wild-type cell. In thiscontext, a wild-type cell is a cell of the same species that does notinclude a mutation in the gene encoding the protein involved in mismatchrepair.

In one embodiment, mismatch repair can be constitutively reduced in thehost cell. Thus, if the cell is a prokaryotic cell, a cell that isdeficient for mismatch repair can have a mutation in one or more nucleicacids encoding mutS, mutH, mutL, uvrD, or dam. The mutS, mutH, mutL,uvrD, or dam protein produced from the mutated gene has a substantiallyreduced (or no) function in mismatch repair. Thus, a correspondingwild-type cell does not have a mutation in the nucleic acid encodingMutS, MutH, MutL, uvrD, or dam, respectively. A cell deficient formismatch repair can also have more than one mutation or the nucleic acidencoding MutS, MutH, MutL, uvrD, or dam, or can have mutations in morethan one of these genes. The mutation can be an insertion, deletion, ora point mutation. Thus, in several specific, non-limiting examples, aprokaryotic cell deficient for mismatch repair has a mutation in anucleic acid encoding MutS (mutS−, or ΔmutS), MutH (mutH− or ΔmutH),MutL (mutL− or ΔmutL), UvrD (uvrD− or ΔuvrD), or Dam (dam− or Δdam), ora combination (e.g. mutS−mutH− (ΔmutSΔmutH), mutS−mutL− (ΔmutSΔmutL),mutH−mutL−(ΔmutHΔmutL), mutH−uvrD−(ΔmutHΔuvrD), etc.).

The homologous nucleic acid (e.g. the single-stranded nucleic acid ordouble-stranded nucleic acid) is introduced into the host cell, thede-repressible promoter is de-repressed, and recombinants are generatedin vivo. Thus, in one specific, non-limiting example, if thede-repressible promoter is P_(L), and the repressor is cI857, the hostcell is treated with heat to induce the expression of Beta (see Copelandet al., Nature Reviews 2:769, 2001, and Ellis et al., Proc. Natl. Acad.Sci. 98:6742-6746, 2001, which are herein incorporated by reference),and optionally Exo and Gam. Generally, the homologous nucleic acid,whether it is a single-stranded nucleic acid or a double-strandednucleic acid, differs from the target nucleic acid by at least onenucleotide, but is sufficiently homologous to undergo recombination withthe target sequence (see above).

Recombinants can be detected by any means known to one of skill in theart. If recombination has occurred in a nucleic acid encoding a marker,such as a nucleic acid encoding a polypeptide involved in antibioticresistance, detection can be performed by drug selection. However,detection can also be performed by direct screening (e.g. colonyhybridization or sequencing). Detection can also be performed bydetecting a label on the nucleic acid (e.g. when DNA includes a DNAadduct or a marker such as biotin).

As has been described (see PCT Publication No. WO 02/14495 A2, hereinincorporated by reference), a single base change has been substituted inthe galK gene and a 3.3 kbp insertion removed from the galK gene usingsingle-stranded oligos. Single-stranded oligos have also been used toprecisely remove five different Tn10 insertions at different places onthe E. coli chromosome. Whereas Exo, Beta, and Gam facilitaterecombination of PCR amplified dsDNA cassettes with flanking homologies,only Beta is required for ssDNA recombination.

Plasmids Conferring Recombineering Functions to Bacterial Host Cells

Disclosed herein are plasmids that can be used to confer recombineeringfunctions to cells, such as prokaryotic cells. These plasmids can beused to confer recombineering functions to a variety of strains of E.coli. In addition, disclosed herein are plasmids that can be used toconfer recombineering functions to other bacteria, including Salmonella,Pseudomonas, Cyanobacteria, and Spirochaetes, amongst others. Thesemobilizable plasmids can be manipulated in vitro and can be used totransform gram negative bacteria. These plasmids include an origin ofreplication specific for the bacterial cell(s) of interest, ade-repressible promoter, and a nucleic acid encoding a single-strandedbinding protein such as Beta. In additional embodiments, the plasmidsinclude a nucleic acid encoding Exo and/or Gam. In one example, theplasmid includes an origin of replication and a lambda genome having DNAencoding functional Beta and optionally Exo, and Gam, or functionalfragments or variants thereof, operably linked to the de-repressiblepromoter (such as, but not limited to, the P_(L) promoter). In oneembodiment, the plasmid is circular, but it can be linearized for someuses. The plasmid can optionally include a selectable marker, such as adrug-resistant cassette (for example, encoding chloramphenicolresistance or ampicillin resistance).

For ease in review, the discussion below refers to the Redrecombineering system, which utilizes the single-stranded bindingprotein Beta, and optionally Gam and Exo. However, one of skill in theart can construct and use the plasmid systems with other single-strandedbinding proteins, such as E. coli RecT, Erf of bacteriophage P22, Rad52of yeast, or ICT8 of Herpes simplex virus.

Generally, the plasmid includes an ori site that allows replication inbacterial host cells, such as gram negative bacterial cells and/or grampositive bacterial cells. The gram negative bacterial cells can be E.coli bacterial cells. However, the ori site can be selected to allowreplication in other bacterial gram negative cells. These gram negativecells include, but are not limited to, Enterobacteriaceae (such asEscherichia, Shigella, Salmonella, Yersinia pseudotuberculosis). Thus,nucleic acids encoding recombineering proteins can also be transferredto Pseudomonas, Acetobacter, Alcaligenes, Bacteroides Amoebobacter,Chromatium, Lamprobacter, Lamprocystis, Thiocapsa, Thiocystis,Thiodictyon, Thiopedia and Thiospirillum, Legionella, Neisseria,Nitrobacter, Nitrospina, Nitrococcus, Nitrosipra, Pseudomonas,Xanthomonas, Zoogloea and Fraturia, Rhizobium, Bradyrhizobium,Azorhizobium, Sinorhizobium Rochalimaea, Ehrlichia, Cowdria, RickettsiaNeorickettsia Spirochaetaceae, Vibrio, Aeromonas, Plesiomonas andPhotobacterium host cells.

The origin of replication can be from any plasmid of interest, and thuscan function in gram negative and/or gram positive cells, and/oreurkaryotes. In one embodiment, the origin of replication (such as theori from plasmid pMB1) functions in a broad host range of gram negativebacterial cells, such as pBBR1, pRK2 or IncQ. However, in otherembodiments, the origin of replication can confer the ability toreplicate in a more limited number of bacterial cells. A conditionalorigin of replication can also be utilized. Conditional origins ofreplication are also known to one of skill in the art. Generally,conditional origins of replication are tight down-regulated in theselected host cells in the absence of a compatible inducing agent, andare strongly induced in the presence of the inducing agent. Theconditional ori, when provided in combination with the compatibleinducing agent, should have sufficient activity to amplify the vectorwithin the host cells. One exemplary conditional ori is oriV, GENBANK™No. L 13843, although the conditional ori could be any ori thatfunctions in the host cell and is normally inactive until exposed to thereplication-inducing agent. Additional conditional origins ofreplication of use are found on plasmids commonly used, such as pBBR1and pSC101. Suitable origins of replication are from the plasmidspSC101, pBBR, pBR322, pUC5, pUC8, pBBR1, RK2, P1, F, amongst others. Inaddition to the ori, the plasmid can include additional plasmidsequences, such as Orf or rep γ that can activate replication. Exemplarynucleic acid sequences of Orf and rep γ are shown in FIGS. 4, 9 and 10.In one embodiment a plasmid can replicate at 32° C. but not at 37° C.

The plasmids include a de-repressible promoter operably linked to anucleic acid encoding a single-stranded binding protein such as Beta.Optionally, the de-repressible promoter also is operably linked to anucleic acid encoding Gam and/or Exo. In one specific, non-limitingexample, the de-repressible promoter is P_(L). The plasmid can alsoinclude one or more of P_(R), O_(L), and O_(R). In one example, theplasmid includes, P_(L) O_(L), and P_(R) O_(R), in the same sequence andorientation as found in phage lambda (λ).

A terminator can be included, such that transcription is terminatedfollowing transcription of bet. If nucleic acids encoding Exo and Gamare included in the plasmid, the terminator can be included 3′ of thesenucleic acid sequences. Terminators are well known in the art, andinclude, but are not limited to, T_(L3) of phage λ. Other terminators ofuse include the T_(INT), T_(L1), T_(L2), T_(R1), T_(R2), T_(6S), T_(OOP)termination signals derived from the bacteriophage lambda, andtermination signals derived from bacterial genes such as the TrpT forthe trp gene of E. coli. These terminators prevent the plasmid originfrom being transcribed by the P_(L) promoter. Thus, in one example,P_(L) is operably linked to nucleic acid sequences encoding Gam, Bet,and Exo, and a terminator, such as T_(L3), is included 3′ of Gam.

Generally, the plasmid encodes a repressor such as cI that binds P_(L).In one embodiment, the repressor is temperature sensitive, such ascI857. In some particular examples, the disclosed plasmids include aninducible promoter upstream of the cI857 gene. The cI gene can alsoinclude an ind1 mutation to prevent spontaneous induction at lowtemperatures.

Optionally, the plasmid can also include a nucleic acid encoding aselectable marker. For example, a nucleic acid can be included thatencodes an enzymatic activity that confers the ability to grow in mediumlacking what would otherwise be an essential nutrient (e.g., the TRP1gene in yeast cells), or that confers resistance (or sensitivity) to anantibiotic or drug upon the cell in which the selectable marker isexpressed. Exemplary markers confer resistance to ampicillin (amp),neomycin (neo), or chloramphenicol (cat). A negative selective markercan also be utilized, such as a nucleic acid encoding the sacB gene(which, if expressed, kills the bacterial host cells grown in mediumcontaining 5% sucrose). In yet another example, a marker is utilized,such as a nucleic acid sequence that encodes an antigenic epitope; cellsthat express the antigenic epitope can be screened and identified by thebinding of an antibody.

In one embodiment, the plasmid includes the following components in 5′to 3′ order: a nucleic acid sequence for an origin of replication; anucleic acid encoding a promoter operably linked to nucleic acidsequence a repressor that specifically binds an operator for ade-repressible promoter, such as the P_(R) promoter; a nucleic acidsequence encoding a selectable marker; a de-repressible promoter that isrepressed by the repressor, such as P_(L) promoter of lambda phage,operably linked to a nucleic acid encoding lambda Beta and a terminator.Transformation of a bacterial cell with the plasmid allows homologousrecombination to occur in the bacterial cell. In another embodiment, theplasmid also includes a nucleic acid encoding an exonuclease operablylinked to the de-repressible promoter. For example, the plasmid caninclude lambda Exo operably linked to the P_(L) promoter. The plasmidcan also include a nucleic acid encoding Gam operably linked to thede-repressible promoter, such as P_(L) promoter. It should be noted thatthe plasmid can include RecE and RecT operably linked to ade-repressible promoter. The plasmid can further include otherregulatory elements, such as P_(R) and O_(R) 5′ of the nucleic acidencoding the selectable marker. Thus, in an additional embodiment, theplasmid comprises O_(L) 3′ of the P_(L) promoter.

In one embodiment, the plasmid includes an origin of replication, anucleic acid encoding a promoter operably linked to a repressor, such ascI, a nucleic acid encoding a plasmid operably linked to the promoter,or to a second promoter, and a nucleic acid encoding Beta operablylinked to the first or the second promoter, or to a third promoter. Anucleic acid encoding Exo and/or Gam can be linked to the first, secondor third promoter. The plasmid does not encode a functional N protein.Exemplary plasmids are pSIM2, pSIM4, pSIM6, pSIM5, pSIM7, pSIM8 andpSIM9. Schematic diagrams of these plasmids, their nucleic acidsequence, and the amino acid sequence of the encoded proteins are shownin FIGS. 4, 7, 8, 9, 10, 11, 12, 13, 14 and 15.

In another embodiment, the plasmid includes an origin of replication anda lambda genome, wherein the lambda genome comprises, in 5′ to 3′ order(1) a repressor that binds the P_(R) promoter; (2) O_(R); (3) a promoteroperably linked to a nucleic acid encoding a heterologous nucleic acidsequence; (4) P_(L); (5) O_(L); (6) a nucleic acid encoding Beta. In oneexample, the plasmid includes an origin of replication and a lambdagenome. As noted above, the origin of replication can be any origin ofreplication that allows replication of the plasmid in the cell ofinterest.

The plasmid can also include a nucleic acid encoding a selectablemarker. The lambda nucleic acid sequence can include a heterologousnucleic acid that encodes a selectable marker. Selectable markers of useare known to one of skill in the art, and are briefly described above.In one embodiment, the selectable marker is included within the rexA oflambda. In another embodiment, the selectable marker is included alongwith rexA and rexB in the lambda nucleic acid sequence.

The lambda can include the phage immunity region and both the O_(L) andO_(R) operators and the main leftward operon under control of the P_(L)promoter, but does not include the major rightward operon encoding theDNA replication genes, the lysis genes and the phage structural genes.Operationally, this means that following prophage induction, theprophage chromosome cannot excise and the cells will not lyse, nor willphage particles be produced.

In yet another embodiment, the lambda nucleic acid includes an N-kildeletion. In one specific, non-limiting example, the lambda genome doesnot encode a functional N (anti-terminator) protein. In another specificexample, the lambda genome includes an N-kil deletion (see FIG. 1) suchthat the lambda does not encode a functional N protein, and such thatthe transcription terminators included in this region between P_(L) andgam are removed. The N-kil deletion leaves intact the Gam gene whose AUGbegins at position 33112 on the lambda DNA sequence and deletes a longerform of the gam gene that has been previous described (Court andOppenheim, pp. 251-277 in Hendrix et al. eds., Lambda II, Cold SpringHarbor Lab Press, (c) 1983, which is incorporated herein by reference).Deletion of the kil gene removes a function that kills bacterial cells,such as E. coli, when expressed.

Thus, the lambda genome can include, in 5′ to 3′ order, a repressor thatbinds the P_(R) promoter at O_(R) and a promoter operably linked to anucleic acid encoding a heterologous nucleic acid sequence, P_(L),O_(L), a nucleic acid encoding Bet. The lambda genome can include anN-kil deletion to remove the genes N through kil. Optionally, additionalsequences are included, such as rep γ or Orf nucleic acid sequences.Nucleic acid sequences encoding Exo and/or Gam can also be included.

A defective λ can be utilized that encodes Red with an intact cIrepression system with various plasmid origins (FIG. 16). In thisprophage, most of the nonessential region of the P_(L) operon has beenremoved, including the toxic kil gene, transcription terminators, andthe anti-termination gene N (FIG. 16A). Other genes are also deleted,such as sieB, ral, ssb and cIII. The rex genes downstream of the cIrepressor gene have been replaced by drug cassettes allowing selectionfor either chloramphenicol (cat) or ampicillin (amp). The P_(L) promoteron these constructs is still regulated by the temperature sensitivecI857 repressor, with O_(L) and O_(R) operators present to ensure thetightest control (Dodd et al., Genes Dev. 15(22):3013-22, 2001; Dodd etal., Genes Dev. 18(3):344-54, 2004). In this system, raising thetemperature and inactivating the repressor directly induces the Redfunctions without the intermediate step of N anti-termination.

A method is disclosed herein for producing plasmids including an originof replication and a lambda phage of interest. Thus, using the plasmidsequences disclosed herein, and the technique of recombineering, manyplasmids can be generated including different origins of replication.For example, primers can be generated that have homology to an origin ofreplication in a plasmid (for example, one of the plasmids schematicallydiagramed in FIGS. 3, 5, 7, 9, 11 and 13), and that have homology to alambda nucleic acid sequence. In one example, the origin of replicationis contained in a pBR322 origin segment, and the plasmid also hashomology at its 5′ end to a lambda nucleic acid sequence. Thus, each oneof the primers in a primer pair includes nucleic acid sequences thatprime DNA synthesis (using amplification techniques, such as PCR) of theorigin of replication, and also include nucleic acid sequences that canpair with (as they are sufficiently homologous to) a lambda nucleicacid. In one embodiment, the set of primers is used to amplify theorigin of replication of interest. Thus, a nucleic acid sequence isamplified that includes the origin of replication, and is flanked bynucleic acid sequences homologous to lambda at each end (homology arms).This nucleic acid, including one homology arm to lambda, an origin ofreplication, and a second homology arm to lambda is then introduced intoa gram negative bacterial cell which contains a lambda prophage insertedinto the chromosomal DNA. The lysogenic lambda DNA present on thebacterial chromosome was pre-induced to produce Beta, Exo and Gam.Optionally, the lysogenic lambda phage includes a heterologous nucleicacid sequence encoding a selectable marker. Once the Red functions oflambda are induced (by de-repressing the P_(L) promoter), and the linearplasmid replicon segment has been introduced (such as byelectroporation), recombination occurs between the linear nucleic acidvector and the lambda prophage. Thus, a plasmid is generated by gaprepair recombination that includes both the origin of replication andthe lambda nucleic acid sequence between the two flanking homologiesincorporated on the linear origin DNA. Following outgrowth, circulargap-repaired plasmids with the origin of replication (such as the pBR322origin of replication) can be isolated using selection for the marker(such as a drug resistance marker) on the prophage and standardmolecular biological techniques, such as a “mini-prep” (see Sambrook etal., Molecular Cloning: a Laboratory Manual, 2nd Ed., (c) 1989).

It should be noted that other plasmid origins like that of pSC101 can beamplified in the same way as that described above for pBR322 with thesame flanking lambda DNA homology using primers. In one example, thelambda DNA is the prophage recombined on the pBR322 origin by gap repairon the plasmid and it is not present on the chromosome. The linearpSC101 with lambda arms is electroporated into cells with the PBR322plasmid carrying lambda Red functions (pSIM2 or pSIM4) which have beenpre-induced to express Red functions. Recombination occurs between thelambda homology on the linear pSC101 and the lambda DNA on the pBR322derivative plasmid. Recombinants are generated that replace the pBR322origin with the pSC101 origin. The recombination mixture is grownovernight and in one embodiment, the plasmid mixture is isolated andintroduced into cells that only allow replication of a specific originof replication of interest. For example, the cells can be polA-defectivecells, which do not allow the replication of pBR322, but allowreplication from alternative origins of replication. In another example,the lambda phage includes a heterologous nucleic acid encoding aselectable marker. Bacterial cells are transformed with plasmids, andthe selection system is utilized to isolate plasmids including an originof replication and the selectable marker.

A method is disclosed herein for producing plasmids including an originof replication and a lambda phage of interest. Thus, using the plasmidsequences disclosed herein, and the technique of recombineering, manyplasmids can be generated including different origins of replication.For example, primers can be generated that have homology to an origin ofreplication in a plasmid (for example, one of the plasmids schematicallydiagramed in FIGS. 3, 5, 7, 9, 11 and 13), and that have homology to alambda nucleic acid sequence. For example, one primer can include asufficient number of consecutive nucleic acids from the 5′ region of anorigin of replication, such as at least 5, at least 10, at least 15, atleast 20 at least 30, at least 50, or at least 100 nucleotides, suchthat the primer can hybridize to the origin of replication. The 5′ endof this primer also includes a sufficient number of consecutive nucleicacids from lambda sequences, such as at least 5, at least 10, at least15, at least 20, at least 30, at least 50, or at least 100 nucleotides,such that the primer can also hybridize to lambda. A second primer caninclude a sufficient number of consecutive nucleic acids from the distalregion of an origin of replication, such as at least 5, at least 10, atleast 15, at least 20, at least 30, at least 50, or at least 100nucleotides, such that the primer can hybridize to the origin ofreplication. The primer also includes a sufficient number of consecutivenucleic acids from lambda sequences at it's 5′ end, such as at leastabout 5, at least about 10, at least about 15, at least about 20, atleast about 30, at least about 50, or at least about 100 nucleotides,such that the 3′ primer can hybridize also to lambda. It should be notedthat about 15 to about 100 nucleotides of a nucleotide sequencehomologous to lambda, such as about 30 to about 100 nucleotides, such asabout 50 to about 100 nucleotides, can be used in each of the first andsecond primers. In one specific example, at least 30 consecutivenucleotides from lambda sequences are utilized, such as 30 to 100consecutive nucleotides from lambda.

These primers are then used in an amplification reaction, such that theorigin of replication is amplified and the product includes a nucleotidesequence homologous to lambda at each end of the amplified product.Homologous recombination (such as “recombineering”) is then used totransfer the new origin of replication such that a plasmid is generatedthat includes a plasmid origin of replication and lambda nucleic acidsequences joined together.

In one example, the origin of replication of interest is a pBR322 originsegment; and the plasmid is constructed that includes the pBR322 originand lambda. Thus, each one of the primers in a primer pair (a firstprimer and a second primer that can be used to amplify a sequence ofinterest) includes nucleic acid sequences that prime DNA synthesis(using amplification techniques, such as PCR) of the pBR322 origin ofreplication, and also include nucleic acid sequences that are homologousto a lambda nucleic acid. The pair of primers is used to amplify theorigin of replication of interest, such as the pBR322 origin. Thus, anucleic acid sequence is amplified that includes the desired origin ofreplication, and is flanked by nucleic acid sequences homologous tolambda at each end (homology arms). This nucleic acid, including onehomology arm to lambda, an origin of replication, and a second homologyarm to lambda is then introduced into a gram negative bacterial cellwhich contains a lambda prophage inserted into the chromosomal DNA (notethat the lambda DNA can also be included on a plasmid, see below). Thelysogenic lambda DNA present on the bacterial chromosome is pre-inducedto produce Beta, Exo and Gam, by de-repressing the de-repressiblepromoter. Optionally, the lysogenic lambda phage includes a heterologousnucleic acid sequence encoding a selectable marker. Once the Redfunctions of lambda are induced (by de-repressing the P_(L) promoter),and the linear plasmid replicon segment has been introduced (such as byelectroporation or transformation), recombination occurs between thelinear nucleic acid and the lambda phage. Thus, a plasmid is generatedby gap repair recombination that includes both the origin of replicationand the lambda nucleic acid sequence between the two flanking homologiesincorporated on the linear origin DNA. Following outgrowth, plasmidswith the origin of replication (such as the pBR322 origin ofreplication) can be isolated using selection for the marker on theprophage and standard molecular biological techniques, such as a“mini-prep” (see Sambrook et al., Molecular Cloning: a LaboratoryManual, 2nd Ed., (c) 1989).

It should be noted that other plasmid origins can be amplified in thesame way as that described above for the origin of pBR322 with the sameflanking lambda DNA homology using primers. For example, the origin ofreplication can be one that functions in a broad host range of gramnegative bacterial cells, such as the origin of replication found onpBBR1, pRK2 or IncQ. However, in other embodiments, the origin ofreplication can confer the ability to replicate in a more limited numberof bacterial cells, such as the origin of replication found in pMB1. Aconditional origin of replication can also be utilized. Conditionalorigins of replication are also known to one of skill in the art, andare described above.

In one example, the origin from plasmid pSC101 is the origin ofreplication. To create a plasmid including this origin of replication,lambda DNA is utilized that is the prophage recombined on a differentorigin, such as a pBR322 origin, by gap-repair (such that the lambda DNAis included on a plasmid and is not present on the chromosome). Thelinear pSC101 with lambda arms is electroporated into cells with aplasmid carrying lambda Red functions, which have been induced.Recombination occurs between the lambda homology on the linear pSC101and the lambda DNA on the pBR322 derivative. Recombinants are generatedthat replace the pBR322 origin with the pSC101 origin. The recombinationmixture is grown overnight and in one embodiment, the mixture ofplasmids is isolated and introduced into cells that only allowreplication of a specific origin of replication of interest. Forexample, the cells can be polA− cells, which do not allow thereplication of pBR322, but allow replication from alternative origins ofreplication.

Growth in a specific cell type is not the only selection system that canbe used to isolate plasmids including an origin of replication andlambda. In another example, the lambda phage also includes aheterologous nucleic acid encoding a selectable marker. This selectablemarker can then be utilized to select the plasmids of interest.

Infective Phage Conferring Recombineering Functions to Bacterial HostCells

Use of a phage with its endogenous regulatory elements can be used toachieve controlled, coordinate expression of the required genes (seeCourt et al., Ann. Rev. Genet. 36:361-88, 2002, herein incorporated byreference). The genome of lambda encodes about 50 genes, all of whichhave been sequenced (see GENBANK™ Accession No. J02459, incorporatedherein by reference, restriction map available at the Fermenta website,and Daniels et al., Appendix II, Complete Annotated Lambda Sequence,Hendrix et al. (eds.), Lambda II, Cold Spring Harbor Press, Cold SpringHarbor, N.Y., pp. 519-676, 1983, herein incorporated by reference intheir entirety). The amino acid sequence of each gene product is alsoknown (GENBANK™ Accession No. J02459 and Daniels et al., supra, bothincorporated by reference in their entirety). It should be noted that aphage in the lysogenic phase, which is integrated into the chromosome ofa bacterial cell is termed a “prophage.” Both prophages and phages areencompassed by the present disclosure.

Disclosed herein is an infectious lambda phage that includes a repressorthat binds a P_(L) promoter, a promoter operably linked to a nucleicacid encoding a heterologous nucleic acid sequence, P_(L), and a nucleicacid encoding Beta operably linked to P_(L), a nucleic acid encoding P,a nucleic acid encoding O, and a nucleic acid encoding cro. At least twoof the nucleic acids encoding P, encoding O, and encoding Cro include anamber codon such that at least two of P, O and Cro proteins are notproduced when the lambda phage is introduced into a suppressor minushost cell. In such suppressor minus cells a lysogenic prophage can formafter infection. The full length O and P gene products allow the phageorigin to replicate from the phage origin. A diagram of the genetic mapof bacteriophage λ is shown in FIG. 2.

In several embodiments, the phage does not include a bacterial orplasmid origin of replication, such as a ColE1 origin of replication,and/or does not include a bacterial initiation site of replication. Forexample, the phage does not include the initiation site (ini) ofColE1replication (see for example, Tomizawa et al., PNAS 74:1865-1869,1977). Generally, for the purposes of this disclosure, unless otherwisestated, a “λ phage” or a “λ prophage” does not include a bacterial orplasmid origin of replication or a bacterial initiation site. Naturallyoccurring non-recombinant phages, including λ phage, do not include abacterial origin of replication.

In one embodiment, the prophage contains the phage immunity region andthe main leftward operon under control of the P_(L) promoter, but ismissing the major rightward operon encoding the DNA replication genes,the lysis cassette, and the structural genes. Operationally, this meansthat following prophage induction, the cells will not lyse, nor willphage particles be produced. In one example, this phage does not includeany origin of replication.

In lambda, the exo, bet and gam genes are clustered in the P_(L) operonand are expressed after induction of the prophage. The cI repressordirectly controls the P_(L) promoter. A temperature sensitive repressormutation, cI857, can be used so that cells transferred to 42° C. arerapidly induced as the repressor is inactivated. This mutant repressorrapidly regains activity upon transfer of the cells to lowertemperatures, so that recombination functions are expressed transientlyfrom P_(L), then shut off completely. Following removal of the repressorby heat induction, the expression of the exo, bet, and gam genes fromP_(L) is initially prevented by transcriptional terminators. Ultimately,λ N function, encoded by the first gene in the pL operon, which isexpressed following de-repression, modifies RNA polymerase to preventtranscription termination (Court and Oppenheim, Lambda II, pp. 251-277,Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1983, incorporatedherein by reference), thereby coordinately activating all the genes inthe pL operon and allowing expression of the recombination functions.

Generally, the phages disclosed herein include Beta operably linked tothe P_(L) promoter. The phages can also include a nucleic acid encodingGam and Exo operably linked to the P_(L) promoter. However, in someexamples, the phages do not encode Exo or Gam. The phage can optionallyinclude repγ, Orf, or both, such as for plasmid replication. In oneexample, the phage encodes its own origin but the replication proteins Oand P are disabled by amber mutations preventing replication.

It is desirable to be able to produce a phage that encodes therecombineering functions (for example, bet alone or bet in combinationwith exo and/or gam) and can infect a cell of interest to transfer therecombineering functions. The recombination functions Exo and Beta of λhave homologs throughout the virus kingdom. These homologs even includethe Herpes virus proteins UL12 and ICP8. Thus, the phage can encodethese proteins instead of Exo and Beta. The cell can be a prokaryotic ora eukaryotic cell. The phage can be λ phage, but is not necessarily λphage. Thus, a bacterial virus or a eurkaryotic virus can be utilized.In several examples, bacteriophage 434, 21 or phi-80 are utilized.

In order to promote homologous recombination once the phage has infectedthe cell of interest, the phage must not enter the lytic cycle in thiscell. Thus, it is desirable to produce a phage (or virus) that (1)transfers recombineering functions to a cell of interest but does notrapidly kill the cell, and (2) in another cell of interest the prophagecan be induced to enter the lytic cycle to produce large quantities ofphage, which in turn can be used to infect additional cells of interest.

In one embodiment, a phage (or prophage) is utilized that includes atleast two amber (stop) mutations. These mutations change a codonnormally encoding an amino acid to a UAG (stop) codon. The ambermutations produce truncated proteins in many wild-type E. coli (such asW3110, DH10B, MG1655, amongst others). The truncated proteins do notretain the function of the full length protein. However, full-lengthproteins can be produced in E. coli that carry amber suppressor tRNAs(such as LE392); in the presence of amber suppressor tRNAs, full-lengthfunctional protein are produced. These are called sup.

The phage (or prophage) includes one amber (UAG stop) mutation in the Por O gene, although amber mutations can be included in both the P andthe O genes. For example, a P amber 80 (also called P sus80 or P80)mutation can be included in the P gene (see Campbell, Virology 14:22-32,1961). In this example, P protein is only produced in a supE carrying E.coli strain. However, any amber mutation can be used in the O and/or theP genes. In one embodiment, the mutation in the P or O gene issuppressed in either a supE or a supF strain.

The phage (or prophage) can also include an amber (UAG stop) mutation incro. For example, the tyrosine at position 26 in the cro gene can bemutated to an amber (UAG stop) codon. In this example, the ambermutation is suppressed in a supF strain such that Cro is produced.Optionally, the phage can also include a temperature sensitive mutation,cI857 in the λ cI repressor gene. Thus, the cI gene, a temperaturesensitive cI protein, and the cro gene, an amber mutation, encodes atruncated defective Cro protein.

In one embodiment, the phage (or prophage) includes an amber mutation inthe P, O or both P and O genes that is suppressed in either a supE or asupF strain. The phage further includes an amber mutation in the Crogene that is suppressed in either a supE or a supF strain. However, theamber mutations differ; one mutation is included that is suppressed in asupE strain and one mutation is included that is suppressed in a supFstrain. For example, the phage can include a mutation in the P gene thatis suppressed by supE and a mutation in cro that is suppressed by supF.The phage can include a mutation in the P gene that is suppressed bysupF and a mutation in cro that is suppressed by supE. The phage caninclude a mutation in the P and the O gene that is suppressed by supFand also includes a mutation in cro that is suppressed by supE.Similarly, the phage can include a mutation in the O gene that issuppressed by supF and a mutation in cro that is suppressed by supE, ora mutation in the O gene that is suppressed by supE and a mutation incro that is suppressed by supF. In this manner, both supE and supF arerequired to induce lysis. Thus, if the phage infects a cell that is supEand supF, the phage will enter the lytic cycle and phage particles willbe produced. When these phage particles infect a cell of interest thatdoes not contain any suppressors, like most “wild-type” strains, thephage does not reproduce and the cell survives. The phage can become alysogen in a cell without a suppressor and repress its P_(L) and P_(R)operons. Thus, the recombineering functions will be transferred to thecell of interest, and homologous recombination can occur inside the cellupon de-repression of the P_(L) promoter at 42° C.

The phage can also include a nucleic acid encoding a selectable marker.Any selectable marker can be utilized, such as a nucleic acid encodingdrug resistance, a nucleic acid encoding an enzyme, or a nucleic acidencoding a detectable label. In one example, the nucleic acid encodes aprotein that confers resistance to an antibiotic. Suitable selectablemarkers encode tetracycline resistance or ampicillin resistance.However, the heterologous nucleic acid can be a nucleic acid thatencodes an enzyme, such as beta-galactosidase, galactokinase ortryptophan synthetase. These markers allow ease of selection of lysogenscarrying the prophage.

Generally, the nucleic acid encoding a selectable marker is insertedinto a phage gene encoding a protein that is not essential for theproduction of phage particles. For example, the nucleic acid encodingthe selectable marker can be introduced to replace the λ rexA and/or λrexB genes, or both. The nucleic acid encoding a selectable marker canalso be introduced in the S gene of phage λ.

Thus, upon introduction of the phage into a host cell including ambersuppressors, phage particles can be produced. However, upon introductionof the phage into a suppressor minus strain, the phage will integrateinto a chromosome of the host cell and exist as a defective prophage.

Thus, a lambda phage (or prophage) is disclosed herein that includes,consists essentially of, or consists of, a temperature sensitive cIrepressor that binds a P_(L) promoter, a promoter operably linked to anucleic acid encoding a heterologous nucleic acid sequence, P_(L)promoter, and a nucleic acid encoding Beta operably linked to P_(L), anucleic acid encoding P, a nucleic acid encoding O, and a nucleic acidencoding Cro, wherein at least two of the nucleic acid encoding P, thenucleic acid encoding O, and the nucleic acid encoding Cro comprise anamber codon such that at least two of P, O, and Cro proteins are notproduced when the lambda phage is introduced into a suppressor minushost cell. The phage (or prophage) can include cI857; the phage (orprophage) does include an origin of replication. In several examples, adefective prophage is also missing the major rightward operon encodingthe DNA replication genes, the lysis cassette, and the structural genes.In an additional example, the phage (or prophage) includes an ambermutation in P and an amber mutation in O, and a tet cassette fortetracycline resistance in the S gene.

Thus, a homologous recombination system can be introduced into cells ofinterest by phage infection. A viable λ plaque-forming phage thatcontains multiple conditional mutations is used to infect a bacterialcell of interest. The phage can optionally include a selectable marker,such as a nucleic acid encoding drug resistance. In specific cell types,the phage will integrate into the nucleic acid of the host cells andwill exist as a prophage. The selectable marker can be used to selectcells of interest that include the phage as a prophage, in order toobtain host cells that express the recombineering functions.

In one example, this phage can be used to introduce recombineeringfunctions into a culture of E. coli containing mixed, completeeukaryotic genomic libraries by infecting the complete librarypopulation with this phage. The introduction of phages into bacterialcells, such as gram negative cells, is well known in the art (seePtashne et al., supra). Thus, lysogens of the entire population(bacterial cells now carrying the integrated phage) can be selectedusing a selectable marker. For example, host cells can be selected thatare tetracycline resistant due to a cassette carried by the infectingphage. In the library strain, the defective prophage construct conveysthe recombination functions and conveys the selectable marker.

Any phage (including but not limited to λ phage) can be engineered toprovide homologous recombination functions (such as Beta, Gam and/orExo) to a host cell. It is known that derivatives of λ phage infect onlya few species of bacteria (such as those related to E. coli K12). Byappropriate engineering of other phages based on the disclosurepresented herein, recombineering functions can be introduced into manyspecies. For example, phages can be engineered for use in Bacillussubtilis or other bacterial cells.

The phages or plasmids disclosed herein can be used to introducerecombineering functions into any host cell of interest. This system canbe used in all types of bacterial cells, including gram negative andgram positive bacterial cells. In this manner, a nucleic acid sequenceof interest can be introduced into a target nucleic acid. Methods aredescribed for example, in published U.S. Patent Publication No.US-2003-0224521-A1, which is incorporated by reference herein.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES

A new in vivo genetic engineering technology, recombineering, developedrecently allows rapid and precise in vivo manipulation of DNA and showsgreat promise for use in functional genomic analysis. It does not relyon restriction sites for cloning, instead uses short regions of homologyand bacteriophage lambda encoded Red proteins (Exo, Bet and Gam) capableof catalyzing recombination between these homologies to link novelcombinations of genes and other genetic elements (see FIG. 1). To beable to use recombineering in different prokaryotic systems, the Redsystem must be transferred to these organisms. A minimal Red expressioncassette on plasmid vectors has been generated and is disclosed herein.These plasmids provide easy mobility in different bacterial backgroundsin addition to tight and coordinated gene regulation.

Example 1 Generation of Plasmid Constructs

Plasmid with phage genes were cloned and generated by a gap repairmechanism which entails the retrieval of Red genes along with the λregulatory elements from a defective lambdoid prophage into linear PCRamplified origin of replication sequences of the plasmids. This methodeliminates standard cloning technology for the phage DNA and importantlythe cloned segment is not replicated in vitro by PCR. Thus, the chancesof extraneous changes occurring in the sequence are reduced.

A prophage lambda derivative was created from the recombineering strainDY330 in which the N through kil genes were deleted using anoligonucleotide of sequence 5′ to 3′ as follows:

-   -   ACGAAACGAAGCATTGGCCGTAAGTGCGATTCCGGATTACTAATCGCCCGGCATTTCGCGGGCGATATTTTCACAGC        (SEQ ID NO: 32; the deletion junction is illustrated, wherein        base 33132 (no underlining, last A in regular text) is next to        base 35445 of phage λ, first base of underlined sequence)),        and deleting lambda published nucleotide sequence from 33131 to        35444 respectively, by recombineering technology. Strain DY330        is temperature sensitive for growth at 42° C. before of kil gene        expression from this prophage. The deletion removes kil,        allowing the DY330 derivative to grow at 42° C. Next the rexAB        coding region was replaced by recombineering with either the CmR        cassette cat or the AmpR cassette amp to create plasmids (termed        pSIM plasmids, see FIG. 16A), selecting for the respective drug        resistance marker.

The genes of this new prophage from pR through exo tL3 were retrievedinto the plasmid ori of pBR322 by recombineering (for example, see FIG.16B). The pBR322 plasmid origin was PCR amplified using primers with 5′homologies to the pR and tL3 regions of lambda (FIG. 16C). The prophagestrain was induced for Red functions and electroporated with the PCR oriproduct. The electroporated cells were diluted into 10 ml of LB andincubated overnight at 32° C. (see FIGS. 3 and 13). The plasmidrecombinant pSIM2 (or pSIM4) was found by transforming strain W3110 andselecting Cm^(R) or Amp^(R), respectively.

The ori segment (from pBR322) was replaced for pSIM2 or pSIM4 with theorigins from the other plasmids such as pSC101, pBBR1 and RK2 usinghomology arms flanking the ori regions made by polymerase chain reaction(see FIG. 16D). Red functions were induced from the pSIM2 or pSIM4containing strains and the homology flanked origin PCR fragments wereelectroporated and the culture diluted into 10 ml of LB for overnightgrowth at 32° C. Mini-preps were made and the plasmid recombinants forCm^(R) or Amp^(R) were selected on a W3110 polA mutant strain which doesnot allow pBR322 origin containing plasmids to replicate. Thus, thepSC101, pBBR1 and RK2 recombinants containing the defective prophagewere selected as recombinant plasmids. Methods used for producing theseplasmids are disclosed in (Constantino and Court, PNAS 100:15748-15753,2003; Court et al., Ann. Rev. Genet. 36:361-388, 2002; Yu et al., PNAS97:5978-5983, 2000; Lee et al., Genomics 73:56-65, 2001; Thomason etal., “Recombineering-Genetic Engineering in Bacteria Using HomologousRecombination,” in Current Protocols in Molecular Biology (Ausbel etal., eds.), John Wiley & Sons, Unit 1.16, 2003, all of which areincorporated herein by reference).

In the plasmid construct most of the non-essential genes in the P_(L)operon have been removed, including the transcription terminators andthe anti-termination gene N. The rex genes of the phage have beenreplaced by a drug marker for selection. The P_(L) promoter is regulatedby the temperature sensitive cI857 repressor and the O_(L) and O_(R)operators are present to ensure tight repressor control. Raising thetemperature from 32° C. to 42° C. inactivates the repressor and inducesthe Red functions from P_(L) without requiring the intermediate step ofN anti-termination. The vector carrying the minimal Red system has theori sequences derived either from pUC or temperature sensitive mutant ofpSC101 or the broad host range plasmid pBBR1 or RK2.

Using the plasmid constructs in E. coli for recombineering,recombination frequencies of ≧10⁴/10⁸ viable cells have been achievedwith PCR products containing drug resistance cassettes, while withsingle-strand oligos recombination frequencies of ≧10⁷/10⁸ viable cellswere observed. The pSC101 based Red system has been successfully appliedin Salmonella to achieve efficient recombination, as shown herein.

Example 2 Exemplary Plasmids

One plasmid/prophage system was generated that has a temperaturesensitive pSC101 replication origin (pSIM5 is Cm^(R); pSIM6 is Amp^(R)).This plasmid catalyzes Red recombination as efficiently as does thedefective prophage in E. coli; the level of unwanted induced backgroundrecombination is also as low as that of the single copy prophage (seeTable 1). A comparison of the chromosomal defective system and theplasmid defective prophage systems described here and the pBAD plasmidexpression system of Datsenko and Wanner (Proc. Natl. Acad. Sci. USA.97:6640-5, 2001) is presented in Table 1.

The same minimal prophage has also been combined with a temperaturesensitive RK2 (pSIM9, Cm^(R)) origin of replication to make anotherbroad host range, low copy number vector. Low copy number reducesundesirable background recombination: a colE1-based plasmid (pUC) originlacking copy number control and carrying the same minimal prophage givesan unacceptably high number of recombinants (about 1,000-fold greaterthan the single copy prophage) in the absence of heat induction (seeTable 1), and places a metabolic load on the host, as evidenced by slowgrowth of cultures carrying these high copy number plasmids (pSIM1 andpSIM2). A broad host range plasmid carrying the pBBR1 origin, pSIM7/8has an intermediate copy number (Table 1). It should be noted that thebroad host range plasmids can be further modified by addition ofmobilization functions that would enable mating between more distantlyrelated species.

Example 3 Recombineering Using galK<>Amp PCR Product for Either E. Colior Typhimurium galK Sequence 1. Linear Drug Cassettes and Single-StrandOligonucleotides Used for Recombineering:

The ampicillin resistant (ApR) cassette amp used to replace the galKgene of E. coli and Salmonella typhimurium was amplified frompBluescript SK (+) (Stratagene) with primers SD3, SD4 and SD5, SD6,respectively (Table 1). The primers contain two parts: a 5′ endhomologous to the flanking regions of galK of E. coli or Salmonella anda 3′ end that primes the cassette for replication (indicated in italics,Table 1).

The ssDNA oligo used for recombineering was supplied by Invitrogen assalt free but otherwise unpurified. The sequence of the 70-mer Oligo 144which corrects the TAG stop codon of E. coli galK gene to a TAC tyrosinecodon is:

(SEQ ID NO: 33) 5′AAGTCGCGGTCGGAACCGTATTGCAGCAGCTT TAC CATCTGCCGCTGGACGGCGCACAAATCGCGCTTAA-3′.

2. Preparation of Cells for Recombineering:

The strains carrying the minimal defective prophage SIMD3/SIMD4 weregrown and induced for Red functions for 15 minutes as in Yu et al.,supra, 2000 Briefly, overnight cultures of SIMD3/SIMD4 grown at 32° C.from isolated colonies were diluted 70-fold in LB medium and grown at32° C. with shaking to an OD₆₀₀=0.4-0.6. Induction was performed on a 15ml culture in a baffled conical flask by placing the flask in a waterbath maintained at 42° C. for 15 minutes under shaking conditions (200revolutions per minute). Immediately after the 15-minute induction, theflask was swirled in ice water slurry to cool for 10 minutes. Anuninduced control culture was also placed into the ice slurry. Thecooled 15 ml culture was centrifuged for 7 minutes at 6,700×g at 4° C.The cell pellet was suspended in 1 ml of ice-cold sterile water followedby addition of another 30 ml of ice-cold water before centrifuging againat 6,700×g for 7 minutes. The supernatant was carefully discarded andthe pellet was suspended in 1 ml of ice-cold sterile water andtransferred to a 1.5-ml eppendorf tube, and was spun for 1 minute at 4°C. at maximum speed in a microfuge. The cell pellet was resuspended in200 μl of ice-cold sterile water and 50 μl of these cells and 100 ng ofPCR product or single-strand oligo were used for each electroporation.After electroporation 1 ml LB was immediately added to theelectroporation mix and the cells were grown at 30° C. either overnight(during making of the Red expression vector) or for 2 hours forrecombineering using both double-stranded DNA or ss oligo before beingdiluted for plating.

3. Screening of Recombinants

Cells were diluted in 1XM9 salt buffer before plating for drugresistance selection. Generally the LA plates containing 30° g/ml ofampicillin or 10 μg/ml of chloramphenicol were used for plating andrecombinants were selected at 32° C. The Gal⁻ phenotype was also testedby streaking colonies on Mac Conkey galactose indicator agar that gavewhite or colorless colonies in contrast to the Red colonies of Gal⁺cells. Gal⁺ recombinant colonies were selected on M63 minimal galactoseplates with biotin and viable cells were counted on LB agar.

4. Results

The results are shown below in Table 2.

TABLE 2 Recombineering with RED Plasmids Using galK <> amp PCR Productfor either E. coli or Salmonella typhimurium galK Sequence Amp^(R)Amp^(R) recombinants per recombinants per Plasmid 10⁸ viable cells 10⁸viable cells Source of RED Origin E. coli S. typhimurium pSIM2 pBR3221.5 × 10⁴ Not Done pSIM5 pSC101ts 2.8 × 10⁴ 9.0 × 10³ pSIM7 PBBR1 2.3 ×10⁴ 9.6 × 10³ pSIM9 RK2ts 4.5 × 10⁴ 1.3 × 10³

The functionality of the Red system has already been demonstrated inSalmonella and recombineering can be used in Salmonella species (see theabove examples). Thus, Red or RecET-like systems will be operative inother gram-negative bacteria closely related to E. coli, such asPseudomonas and Streptomyces species, or Vibrio and Shigella. It shouldbe noted that dsDNA viruses other than λ encode Red-like SynExo twocomponent recombinases (see Vellani and Myers, J Bacteriol. 185:2465-74,2003; Reuven et al., J. Virol. 77:7425-33, 2003, Mikhailov et al., J.Virol. 77:2436-44, 2003); such recombinases are likely to catalyzeefficient and accurate recombination in their particular host, and couldbe substituted for lambda Red functions. Currently, there are threewell-studied families of exonucleases (λ Exo, RecE and ABC2-modifiedRecBCD) and five families of synaptases (Beta, RecT, ERF, ICP8 andLEF-3), identified by BLAST searches, ultrastructural analysis andenzymology. Thus, there is a superfamily of Beta single-strand annealingproteins with members widespread throughout the prokaryotic world.Indeed, members of the Red Beta family have been in many bacteria,including Borrelia, Listeria and Streptococcus. The Bacillus phage SPP1Chu/gp35 SynExo recombinase (Vellani and Myers, supra, 2003) could alsobe used for recombineering in Bacillus bacterial cells. Phage functionsthat co-evolved with the host bacteria are optimized to maintainallele-specific interactions with host proteins likely to facilitatehigh efficiency and high fidelity recombination. Without being bound bytheory, for optimal recombineering, it is believed that the nuclease andsynaptase pairs should be evolutionary partners.

The λ Gam protein specifically inhibits the E. coli RecBCD and SbcCDnucleases, and BLAST analysis suggests that it is less widelydistributed than the Red and RecET functions, as it is present in onlysome pathogenic strains of E. coli, Shigella, and Salmonella prophages.While RecBCD-like ExoV enzymes are found in many gram-negative bacterialspecies, the gram-positive bacteria often contain a two-subunit form ofthis enzyme (AddAB or RexAB). Given the widespread distribution of ExoVactivity in bacteria, there may be functional analogs of Gam in otherphages. If analogs to ExoV and the SbcCD nucleases are not present inthe organism of interest, λ Gam is likely to be ineffective, and othernon-RecBCD-like nucleases may degrade introduced linear DNA even in thepresence of Gam. Some phages have proteins that protect linear DNA fromdegradation, and some of these could, like λ Gam, protect dsDNA whilestill allowing its participation in recombination reactions. It shouldbe noted that not all phage-encoded nuclease inhibitors will be usefulin this context, since some (i.e. Mu Gam and T4 gp2) act by apparentlybinding DNA ends, protecting them from degradation, but also making themunavailable to participate in recombination.

Example 4

Generation of a Phage

The sequences of the rexAB genes and the S gene of lambda are shown inFIGS. 17 and 19; the tetracycline cassette is inserted to replace thesegenes in lambda (see FIGS. 18 and 20).

The first phage used was lambda cI857 ind1 rexAB<>tetRA Cro Tyr26 TAGPam80=Gln60 TAG. A second phage is created that is identical to thefirst except that rexAB will be intact and the S gene will be replacedwith tetRA [S<>tetRA] (see FIG. 18).

Lambda cI857, used for the generation of the multiply mutant phages (aphage with more than one mutation in its genome), had the cI857 allelechange and the ind1 allele, which is indicated in the cI gene annotationof that sequence (see Ptashne, M., “A Genetic Switch,” Third Edition,phage Lambda Revised, Cold Spring Harbor Lab, New York, ISBN No.0-8769-716, 2004). The prophage in strains DY329, DY330, DY331, DY378include the cI857 allele but are ind+ (these strains do not carry theind1 mutation), while the prophage in strain DY380 carries both thecI857 and ind1 mutant alleles.

The multiply mutant lambda phage was further changed by adding twoadditional point mutations, both of which were amber mutations. Theamber mutations were introduced into the phage so that the phage will bedefective for killing with a 15 minute induction at 42° C. (because ofthe P amber 80 mutation, see below) and will be constitutive for pLexpression (because of the Cro amber mutation, see below) as a prophagein a particular E. coli strain. The amber mutations cause defectiveproteins in E. coli including W3110, DH10B, MG1655 and others. In thismanner, a pair of amber mutations was inserted that are suppressed bytwo different suppressor tRNAs for functional expression of both geneproducts.

Both of these suppressor tRNA mutations are present in LE392 strain. Inthis host (or similar hosts with both suppressors) this phage madeplaques and could be propagated lytically to make high titer lysates (>1billion phage/ml). These lysates could be used to infect and lysogenizethe DH10B like strains, which have no suppressor tRNA (and are calledsup zero or sup minus). In these sup zero strains, the phages aredefective (and have an effect similar to the defective prophages inDY329 and DY380). However, it should be noted that a substantialdifference exists between the presently generated phage and theprophages DY329 and DY380: high titer phage lysates can be made usingthe phages containing the amber mutants by infecting sup⁺ hosts likeLE392.

The amber mutation in Cro changes a tyrosine codon at position 26 in thecro gene to a UAG amber codon. This amber mutation is suppressed only bythe supF tyr tRNA (see Oppenheim et al., Virology 319:185-189, 2004,incorporated by reference herein, which describes methods of use ingenerating amber mutants in lambda). The amber mutation in P is the Psus80 mutation (see Campbell, Virology 14:22-32, 1961, which isincorporated by reference herein). This mutation is also called P80 or Pamber80. This mutation was added to the cI857 ind1 cro amber phage usingthe Oppenheim method of mutagenesis (see Oppenheim et al., op. cit.,incorporated by reference). These mutations were detected by being ableto form plaques on LE392 but not a supF only strain (because the P amberis not suppressed by supF; it needs supE to allow P protein to be made).This phage does not grow on a supE only strain like C600 because of thecro amber mutation.

It should be noted that other combinations of different ambers could beused in cro or P. Alternatively, an amber mutation in O could beproduced, instead of P, as a prophage that includes an amber mutation inO is also replication defective. Thus, a prophage including an ambermutation in O and an amber mutation in cro could also be lytic in theLE392 strain, but not in a sup minus strain of bacteria.

Once the amber mutations were introduced into the phage, the tetR genewas PCR amplified with flanking homologies to the rexAB genes. FIG. 17shows the sequence of the rexAB genes. Using recombineering, these tetgenes were crossed into the amber mutant phage in the rex region asshown in FIG. 18. In addition, another phage was recombined with tetRAreplacing the S gene (see FIG. 19) of the amber phage. Thus, a similarcross (as with rex) was done by recombineering using S flankinghomologies to insert tetRA in place of S gene in the phage carrying theamber mutants (see FIG. 20). When rexAB is replaced the phage still madeplaques on LE392 but not on DH10B. However, Tet resistant lysogens canbe formed in DH10B (or other strains on which plaques do not form). Thephage enters the lysogenic cycle and integrates its DNA in the bacterialchromosome at attB. When S is replaced by tetRA then the phage becomesdefective for plaque formation in all strains because S is needed tomake a plaque. However, S is not needed to produce phage in bacterialcells. Thus, the recombineering cross to create the S<>tetRA mutant wasitself used to infect LE392 and TetR lysogens were selected in thesuppressor strain.

These lambda cI857 ind1 Cro amber Pamber S<>tetRA lysogens of LE392 canbe induced by growing a culture in LB broth until OD 600 of 0.4 at 32°C. and then shifting to 42° C. for 15 minutes and back to 39° C. for 2hours with shaking in a water bath. During these two hours, phageproduction progressed in LE392 and the cells did not lyse (because the Sgene is needed for lysis). After two hours the cells were concentratedby centrifugation and suspended in a small volume of buffer (Tris 0.01M,Mg⁺⁺ 0.01M) and treated with chlorophorm, which caused lysis and releaseof large numbers of phage (>10 billion/ml). This pure preparation ofphage can be used to infect DH10B or other suppressor defective E. coliselecting for TetR lysogens at 32° C.

This strategy allows (with either the rex or S phage) the selection ofTetR lysogens in various hosts, including recA defective hosts or otherrecombination defective strains. This strategy also allows largecultures of non-lysogenic strains. For example, a BAC library of humangenomic clones or another such library can be infected and converted toTetR, being lysogenic for these defective lambda phages. These defectiveprophages can be used to induce recombineering in any cell of interest.Temperate phages other than phage λ can be changed to ind⁻-like as shownby Friedman et al. Such ind⁻ phages can then be converted to carry atemperature sensitive repressor like cI857 (see Tyler and Friedmen, J.Bacteriol. 186:7670-9, 2004).

Mutations can be created in phage, including bacteriophage λ, usingrecombineering. Briefly, E. coli harboring a defective λ prophage isinfected with the phage to be engineered. The partial prophage carriesthe P_(L) operon under control of the cI857 temperature sensitiverepressor. The lysogen is induced to express the Red functions (Beta,Gam and/or Exo), the induced cells are made competent forelectroporation, and the PCR produce of oligonucleotide is introduced bya standard method of introducing nucleic acids into bacterial cells,such as electroporation. Following electroporation, a phage lysate ismade from the electroporation mix.

Thus, as disclosed herein, mutations can be created in phage, includingbacteriophage λ, using recombineering. Briefly, E. coli harboring adefective λ prophage is infected with the phage to be engineered. Thepartial prophage carries the P_(L) operon under control of the cI857temperature sensitive repressor. The lysogen is induced to express theRed functions (Beta, Gam and/or Exo), the induced cells are madecompetent for electroporation, and the PCR product of oligonucleotide isintroduced by a standard method of introducing nucleic acids intobacterial cells, such as electroporation. Following electroporation, aphage lysate is made from the electroporation mix. (see Oppenheim etal., Virology 319:185-189, 2004, which is herein incorporated byreference in its entirety).

Example 5 Generation of Phages

Phages were generated that include suppressible mutations by introducingUAG termination codons in the essential λ genes O, P, Q, (Oppenheim etal., Virology 319:185-189, 2004, which is herein incorporated byreference in its entirety) S, and E. The target phage λ cII68 acquiredthese amber mutations at a frequency of 1-3% in a cross with70-nucleotide long single-stranded oligos with the UAG codon at thecenter of the oligonucleotide. Amber mutants were easily identified ascloudy plaques with a double-layer bacterial lawn (see Campbell, GeneticStructure. In: Hershey (ed), The Bacteriophage Lambda, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., pp 13-44, 1971;Oppenheim et al., supra). The lower layer contains the restrictive hostW3110 and the top layer contains the infected supF suppressor host LE392cells and form cloudy plaques (because W3110 cells in the lower layergrow to confluence and remain unlysed by the amber mutant phage).

Previous studies of λ Cro function were based primarily on the use ofone missense mutant, cro27. The phage cI857 cro27 forms clear plaques at37° C. but cannot form plaques at either 32° C. or 42° C. (Eisen, H.,and Ptashne, M., “Regulation of repressor synthesis,” A. D., p. 239-245,1971). The Cro protein contains three tyrosine residues, and eachtyrosine codon (70-base oligo used) was independently replaced with UAG.Screening plaques at 42° C. in a double layer, approximately 2% of totalplaques were cloudy. On LE392, the resultant mutants grow at 32° C., 37°C. and 42° C., but on W3110 they form plaques only at 37° C. An80-nucleotide oligonucleotide was used to generate a 326-bp deletion ofthe cII gene in λ c⁺. This ss-oligo provides 40 bases of homology ateach end of the segment to be deleted. λ c⁺ normally forms turbidplaques. Clear plaque recombinants were found at a frequency of 2%.Sequencing showed that the resulting clear mutant phage carried adeletion exactly corresponding to the original design. This deletionfuses the cII translation initiation condon to the downstream O gene,creating a phage with O at the normal cII location. It should be notedthat, using recombineering, deletions as large as 5 kb have beengenerated with oligonucleotides on the E. coli chromosome with goodefficiency.

The phage λ rexA and rexB genes were replaced precisely with a bla geneconferring ampicillin resistance. The bla gene was first amplified byPCR using primers with 5′ homology to the flanking regions of the rexABgenes; the PCR product was then targeted to the λ chromosome withrecombineering. A phage lysate was grown from the electroporation mixand used to form lysogens. AmpR lysogens were selected and thereplacement of the rexAB genes by the bla gene in such lysogens wasconfirmed by PCR analysis (Yu et al., supra, 2000) and by the ability ofthe recombinant lysogens to plate T4rII mutant phage (Benzer, Proc.Natl. Acad. Sci. U.S.A. 41:344-354, 1955). Using appropriate PCR primersand the gene SOEing technique (Horton et al., Biotechniques 8(5):528-535, 1990), a linear DNA product was created containing anintact copy of the wild-type λ P gene adjoining a precise deletion ofthe entire ren gene but with homology beyond ren in the ninR region ofthe phage. The construct was targeted to an infecting Pam80 phage; P⁺recombinants were selected and screened for the ren deletion. P⁺recombinants were obtained at a frequency of 2%; 20% of these had thedeletion.

1. Analysis of Mutations Arising from the Use of Oligonucleotides inRecombineering.

It has been demonstrated that recombineering provides an efficient wayto manipulate the bacteriophage genome. However, it was found thatsometimes oligonucleotide recombination has associated unwantedmutations. To understand the origin and nature of these unwantedmutants, a protocol was designed to score for both true recombinants andunwanted changes. Phage λ cI857 carries a temperature-sensitive mutationin repressor; thus, the phage forms clear plaques at 37° C. and turbidplaques at 30° C. (Sussman and Jacob, C. R. Acad. Sci. (Paris)254:1517-1519, 1962). Two complementary oligonucleotides were designedthat were 82 residues long, with wild-type repressor gene sequence thatcould generate wild-type λ recombinants in a cross with λ cI857 (FIG.1). These oligos cover about 1/10 of the cI coding region and arecentered on the cI857 allele. The recombinant lysate was diluted andplated on W3110 at either 37° C. or 32° C. At 37° C., λ c⁺ recombinantsform turbid plaques. At 32° C., both parent and recombinant should formturbid plaques. When plaques from the recombineering cross were grown at37° C., most were clear, however, 4-13% were turbid as expected ofwild-type recombinants (Table 1). When the recombinant lysate was platedat 32° C., most plaques were turbid as expected, however, a significantproportion, 0.5-2%, was clear. This number is 10-40 times higher thanthe spontaneous frequency of clear plaques (approximately 0.05%) foundin lysates prepared the same way but without the addition ofoligonucleotide or with the addition of an oligonucleotide lackinghomology.

To understand the source of the unwanted clear mutations, clear andturbid recombinants were purified and sequenced their cI gene. Fourteenturbid λ cI⁺ recombinants isolated at 37° C. had all been corrected forthe cI857 mutation without additional mutations. However, all clearplaques identified at 32° C. contained other mutations of cI. Thesemutations were about equally produced by the two oligonucleotides.Twenty-four or twenty-five of those sequenced had mutations in theregion covered by the ss-oligo. Among these 24 mutants, 22 had alsoconverted the cI857 allele to wild-type. One of these 22 mutants was aGC to TA transversion, the rest were deletions of one or more bases ofthe cI sequence. The one change outside of the oligo region was a GC toTA transversion that retained the cI857 allele and possibly arosespontaneously.

To demonstrate that these mutations were not specific to cI857 or to theoligo sequence, the experiment was repeated using wild-type λ cI⁺ andcomplementary ss-oligos from a different region of the cI gene in across. These oligonucleotides carried a single silent AT to GC change.As before, clear plaques were found in the lysate followingrecombineering. The DNA from 16 clear plaques was sequenced. Fifteencarried the silent mutation indicating that they had undergonerecombineering. Nine had a single base pair deletion, three had longerdeletions, one mutant had an added AT base pair, one showed a CG to TAtransition, and one had a GC to AT base substitution mutation locatedoutside the region covered by the ss-oligo. The one mutant lacking thesignature change had a CG to TA transition outside the region covered bythe ss-oligo and may have been a spontaneous clear mutant.

Exp. # Host Oligo λ c⁺ % λ cI⁻ % 1 DY433 W 6 2 2 HME31 W 6 1.5 3 HME31 C13 2 4 HME31 W 4 0.5 recA 5 HME31 — <0.05 <0.05 5 HME31 #100 <0.05 <0.056 HME31 C* 3.2 0.1

The results presented above suggest that most of the mutations wereintroduced during synthesis of the single stranded oligonucleotides(ss-oligos). Based on the results and chemistry of synthesis, one wouldexpect that at each position of the oligonucleotide there would be anequal chance of not incorporating the added base (Hecker and Rill,Biotechniques 24(2):256-260 1998; Temsamani et al., Nucleic AcidsResearch 23(11):1841-1844 1995). Examination of the sequence changesamong the frameshifts show that they cluster toward the center of thess-oligo. The terminal regions lack mutations, suggesting that completebase pairing at the termini may be important for efficient annealing tothe phage DNA.

To reduce the frequency of frameshift mutations, the ss-oligos werefurther purified. Purification by HPLC did not reduce the mutationfrequency (data not shown) probably because HPLC does not efficientlyseparate oligos of this length, whereas PAGE-purified oligonucleotidesyielded efficient recombineering with fewer frameshifts (see abovetable). This result supports the notion that base deletions originatingduring chemical synthesis of the oligonucleotides are responsible forgenerating mutations. Single base frameshift deletions occur rarely asspontaneous mutations (Schaaper and Dunn, Genetics 129(2):317-326,1991). In the above examples, deletion mutations formed usually alsocarried the designed change present on the ss-oligo, suggesting that theframeshifts were conferred by the synthetic ss-oligo. Thus, theexperimental approach described herein provides a simple and sensitiveassay for oligonucleotide quality. Recombineering with unpurifiedsynthetic oligonucleotides could also be used to provide an efficientway to introduce random single base deletions at specific sites in genesor regulatory regions. The results do not suggest that the act ofrecombineering causes random mutagenesis.

When recombineering with the bacterial chromosome, one of twocomplementary ss-oligos gives more recombinants (Ellis et al., supra,2001; Zhang et al., Nature Genetics 20:123-128, 2003). This strand biasdepends upon the direction of replication through the recombining regionwith the lagging strand being the more recombinogenic. In the phagecrosses, both complementary oligos were equally efficient in promotingrecombination at λ cI. Without being bound by theory, this can be due tothe rolling circle mode of phage DNA replication, which can roll ineither direction (Takahashi, Mol. Gen. Genet. 142(2):137-153, 1975).Thus, replication forms pass through cI in both directions and neitherstrand is exclusively leading or lagging.

In the cross with λ cI857, mottled plaques were observed at 37° C.,which suggested that the λ DNA was packaged with a heteroduplex allelein cI (Huisman and Fox, Genetics 112(3):409-420, 1986). Six independentmottled plaques were purified; they gave rise to a mixture of turbid andclear plaques. Sequence analysis showed that in all cases the turbidplaques had incorporated the wild-type allele, whereas the clear plaquesretained the original cI857 mutation, indicating that theoligonucleotide paired with the phage chromosome and was incorporatedwithout mismatch correction. These heterozygous phages are generated inrecA mutant crosses, which suggests that the ss-oligo is annealed byBeta protein to single-strand gaps at the replication fork (Court etal., Annu. Rev. Genet. 36:361-388, 2003; Stahl et al., Genetics147(3):961-977, 1997).

2. Materials and Methods

a. Creating Mutations with Recombineering.

The strains used for recombineering carry a defective λ prophagecontaining the p_(L) operon under control of the temperature-sensitiverepressor cI857. The genotype of one commonly used strain, DY330, isW3110 ΔlacU169 gal490 pglΔ8λcI857 Δ(cro-bioA). The strain of choice isgrown in a shaking water bath at 32° C. in LB with 0.4% maltose tomid-exponential phase, A₆₀₀ 0.4-0.6 (30 ml is adequate for severalrecombineering reactions). The culture is harvested by centrifugationand resuspended in 1 ml TM (10 mM Tris base, 10 mM MgSO₄, pH 7.4). Thephage to be engineered is added at a multiplicity of infection of 1-3phages/cell (assume a cell density of approximately 1×10⁸/ml beforeconcentration) and allowed to adsorb at room temperature for 15 minutes(this step would need modification for other phages, i.e., adsorption onice). Meanwhile, two flasks with 5-ml broth are prewarmed to 32 and 42°C. in separate shaking water baths. The infected culture is divided andhalf-inoculated into each flask; the cultures are incubated anadditional 15 minutes. The 42° C. heat pulse induces prophage functions;the 32° C. uninduced culture is a control. After induction, the flasksare well chilled in an ice water bath and the cells transferred tochilled 35-ml centrifuge tubes and harvested by centrifugation atapproximately 6500×g for 7 minutes. The cells are washed once with 30-mlice-cold sterile water; the pellet is quickly resuspended in 1-mlice-cold sterile water and pelleted briefly (30 seconds) in arefrigerated microfuge. The pellet is resuspended in 200-μl cold sterilewater and 50-100 μl aliquots are used for electroporation with 100-150ng PCR product or 10-100 ng oligonucleotide. A BioRad E. coli GenePulser was used set at 1.8 mV and 0.1-cm cuvettes. Electroporated cellsare diluted into 5 ml 39° C. LB medium and incubated to allow completionof the lytic cycle. The resulting phage lysate is diluted and titteredon appropriate bacterial to obtain single plaques (for more details, seeThompson et al., Current Protocols in Mol. Bio. 1.16.1-1.16.16, 2003).

b. Oligonucleotides.

The oligonucleotides were purchased from Invitrogen without additionalpurification. The purified oligonucleotide was subjected toelectrophoresis in a 15% PAGE-Urea gel, excised from the gel withoutdirect UV irradiation and eluted using the Elutrap electro-separationsystem (Schleicher and Schuell). The size-purified oligonucleotide wasthen precipitated with isopropanol, washed with ethanol, dried andstored at −20° C.

Example 6 Use of Single-Strand Oligo Recombineering in Salmonella

Wild-type galK Salmonella does not grow on minimal medium with glycerolas carbon source when 2-deoxygalactose is also present. This is due tothe galactokinase function of galK⁺, which converts 2-deoxygalactose toa toxic compound for the cell causing death. Forms of Salmonella thatare galK− can grow on glycerol with 2-deoxygalactose. Theoligonucleotide sequence of the Salmonella typhimurium galK gene isshown with the changes incorporated to make it Gal-(SEQ ID NO: 21). TheS. typhimurium gal .GTGTTCA oligo has the sequence set forth below thatis 70 bases in length:

(SEQ ID NO: 34) AAGTGGCGGTGGGCACCGTCTTCCAGCAGCTTTAGTGTTCACCGCTGGACGGCGCGCAAATTGCGCTCAAThis oligonucleotide was used to mutate the galK gene to Gal−. Thesequence shown as SEQ ID NO: 34 has the 7 bases underlined changed fromthe wild-type sequence.

The Salmonella typhimurium (St)144 oligonucleotide has the sequence setforth below:

(SEQ ID NO: 35) AAGTGGCGGTGGGCACCGTCTTCCAGCAGCTTTACCACCTGCCGCTGGACGGCGCGCAAATTGCGCTCAA

The St 144 oligonucleotide was used to correct the mutation, and makethe cells Gal+. It should be noticed that this oligo creates thewild-type galK sequence with the exception of the TAT tyrosine codon,which became TAC tryrosine in the Gal⁺ recombinant.

These oligonucleotides were introduced into S. typhimurium cellstransformed with plasmid pSIM5. Red functions were induced by a 15minute temperature shift to 42° C. Recombinants with the S. typhimurium.galK.GTGTTCA oligo become galK− and survive on 2-deoxygalactose, butcannot use galactose as the only carbon source. GalK⁺ recombinantsproduced using the 144 oligonucleotide grow on galactose as sole carbonsource and were selected in minimal galactose agar. These Gal⁺ strainswere produced using recombineering with the St 144 oligo.

The new galK+ strain produced by recombineering differed from wild-typeat the one base of the TAT codon position 144 where the recombinant isTAC. The frequency of going from galK+ to galK−, or from galK− togalK+(in the two recombineering crosses with oligos S. typhimuriumgalK.GTGTTCA and S. typhimurium (St)144) was 5% in each case. Theseresults demonstrate that the plasmids disclosed herein can be used tointroduce recombineering functions into S. typhimurium.

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. A plasmid, comprising an origin of replication (ori), a nucleic acidencoding a selectable marker, a nucleic acid encoding a promoteroperably linked to a nucleic acid sequence encoding a repressor thatspecifically binds a P_(L) promoter of lambda phage, and a lambda genomecomprising the P_(L) promoter of lambda phage, and a nucleic acidencoding Beta operably linked to a promoter and a terminator, whereinthe lambda genome contains an N deletion and a kil deletion, and whereinbacterial host cells transformed with the plasmid can perform homologousrecombination.
 2. The plasmid of claim 1, wherein the ori is selectedfrom the group consisting of pSC101, colE1, pBR322, pBBR1 and RK2
 3. Theplasmid of claim 1, wherein the selectable marker replaces a rexABcoding region in the lambda genome.
 4. The plasmid of claim 1, whereinthe on allows replication of the plasmid in Salmonella typhimurium,Escherichia coli, or both.
 5. The plasmid of claim 1, further encodingExo or Gam.
 6. The plasmid of claim 1, wherein the lambda genome encodesrexA and rexB.
 7. The plasmid of claim 1, wherein the terminator isT_(L3), T_(INT), L_(L1), T_(L2), T_(R1), T_(R2), T_(6S), or T_(OOP) oflambda phage.
 8. The plasmid of claim 7, wherein the terminator isT_(L3), and wherein the selectable marker is chloramphenicol resistance.9. The plasmid of claim 1, wherein the repressor is cI857.
 10. Theplasmid of claim 1, comprising in 5′ to 3′ order (i) the origin ofreplication (2) the nucleic acid encoding the promoter operably linkedto a nucleic acid encoding a repressor that binds the P_(L) promoter;(3) the promoter operably linked to a nucleic acid encoding Beta. 11.The plasmid of claim 1, wherein the on is pBR322 or pSC101.
 12. Achimeric plasmid, comprising an origin of replication (ori) selectedfrom the group consisting of pSC101, colE1, pBBR1 and RK2; and λ phagenucleic acid, wherein the λ phage nucleic acid comprises a) a nucleicacid encoding Beta operably linked to P_(L), b) O_(L) and O_(R); c) anucleic acid encoding cI857; d) an N deletion, e) a kil deletion; and e)a nucleic acid encoding a selectable marker; and wherein bacterial hostcells transformed with the plasmid can perform homologous recombination.13. The chimeric plasmid of claim 12, wherein the chimeric plasmid doesnot comprise the N-kil genes of lambda phage.
 14. The plasmid of claim13, further comprising nucleic acid encoding Orf and repy.
 15. A methodof producing a bacterial host cell that can perform homologousrecombination, comprising introducing the plasmid of claim 1 into thebacterial host cell, thereby rendering the bacterial cell capable ofperforming homologous recombination.
 16. The method of claim 15, whereinthe host cell is a gram negative bacterial cell.
 17. The method of claim16, wherein the gram negative bacterial cell is an Escherichia colicell.
 18. A plasmid, comprising an origin of replication selected fromthe group consisting of pSC101, pBR322, colE1, pBBR1 and RK2, a nucleicacid encoding a selectable marker, a nucleic acid encoding a promoteroperably linked to a nucleic acid sequence encoding a repressor thatspecifically binds a P_(L) promoter of lambda phage, and a lambda genomecomprising the P_(L) promoter of lambda phage operably linked to anucleic acid encoding Beta and a terminator 3′ of the nucleic acidencoding Beta.
 19. A method of producing a bacterial host cell that canperform homologous recombination, comprising introducing the plasmid ofclaim 12 into the bacterial host cell, thereby rendering the bacterialcell capable of performing homologous recombination.
 20. A method ofproducing a bacterial host cell that can perform homologousrecombination, comprising introducing the plasmid of claim 18 into thebacterial host cell, thereby rendering the bacterial cell capable ofperforming homologous recombination.
 21. The method of claim 20, whereinthe bacterial host cell is a gram negative bacterial host cell.